Dr. Rob Dillon, Coordinator





Tuesday, July 12, 2016

Pleurocera clavaeformis in the Mobile Basin?


This is the final installment of my five-part series [1] reviewing an excellent recent paper by Nathan Whelan and Ellen Strong [2] on DNA sequence variation within and among an assortment of pleurocerid populations sampled from North Alabama.  The most important contributions made by Whelan & Strong will turn out to be their large and fine data set on mitochondrial superheterogeneity, and their demonstration that no morphological characters seem to correlate with it, putting to rest the hypothesis of cryptic speciation.  Their paper also sheds new light on evolutionary relationships among the pleurocerid populations of the Mobile and Tennessee drainages, if one is able to contextualize the information they have developed.  Such will be our business in the essay that follows.

Whelan and Strong sampled five populations of Leptoxis picta (which they identified as L. “ampla”) from tributaries of the Alabama River draining south into the Mobile Basin and one population of the widespread and well-studied Leptoxis praerosa sampled from a tributary of the Tennessee River, draining west.  Despite the strikingly high incidence of mitochondrial superheterogeneity demonstrated by the five picta populations, the praerosa population remained genetically distinctive, demonstrating about 13% CO1 sequence divergence from modal picta.  This observation dovetailed neatly with the (nuclear) histone H3 sequence data also developed by Whelan & Strong, and with a much larger (9 locus, 11 population) allozyme data set published by Chuck Lydeard and myself in 1998 [3], all of which combined to strongly reinforce the validity of the specific distinction between L. picta populations inhabiting the Mobile Basin and L. praerosa inhabiting the Tennessee.

But such did not seem to be the case with the five populations Whelan & Strong identified as “Pleurocera prasinata” from tributaries of the Mobile Basin, when compared with the population they identified as “Pleurocera pyrenella” from a tributary of the Tennessee.  As we brought our essay of 3May to a close, we noticed that the histone H3 data did not return any evidence of a genetic distinction between any of the six Pleurocera populations whatsoever.


And indeed the larger dataset developed by Whelan & Strong on CO1 sequence divergence did not demonstrate a distinction between any of the six North Alabama Pleurocera populations either.  The gene tree above shows that the modal CO1 haplotype sequenced from the Tennessee “pyrenella” population (“clade 6”) was just 2% different from both the modal prasinata clade 8 and the first-runner-up prasinata clade 7 [4].  But the gene tree below shows that CO1 sequence divergence within the Tennessee “pyrenella” population ranged up to 8% [5].


Nor indeed is there any significant morphological distinction among Whelan & Strong’s six Pleurocera populations.  Last month we reviewed the general topic of ecophenotypic plasticity in shell morphology, as it applies to the Pleurocera populations of North Alabama.  It was our strong impression that the Tennessee drainage Pleurocera population sampled by Whelan & Strong, referred by them to “Pleurocera pyrenella,” was misidentified.  In fact, Whelan & Strong’s Figure 7, reproduced in last month’s post, suggested that all six North Alabama Pleurocera populations are morphologically indistinguishable from Pleurocera clavaeformis.

Now here is the second-most amazing revelation to come from the Whelan & Strong’s remarkable data set [6].  In order to explain the amazingness of this revelation, however, I’ll need to digress a bit into the basic mechanics of the NCBI GenBank.  And backtrack to 2011.  So bear with me.

Among the most useful and powerful tools on the GenBank menu is “BLAST,” the “Basic Local Alignment Search Tool.”  A user can call up any sequence from the database, say for example KT164088, the first CO1 sequence Whelan & Strong listed in their Table 1 for modal CO1 clade 8 of “Pleurocera prasinata.”  And then simply click the BLAST button (optimized for more dissimilar sequences), and wait a minute or two.  And the BLAST tool will return the (default) 100 sequences in GenBank most similar to KT164088.  Amazing.  I never thought I would live to see the day.

So W&S uploaded all 5 x 20 = 100 of their “prasinata” CO1 sequences to GenBank, plus all 20 of their “pyrenella” CO1 sequences, for a total of 120 CO1 sequences from North Alabama Pleurocera [7].  You might think that if you simply picked a representative sequence from the modal “prasinata” CO1 cluster and hit the BLAST button, the search tool would return 100 other North Alabama Pleurocera sequences, wouldn’t you?  But this is not the case.  Down toward the bottom of the big list you will get if you perform the experiment I have outlined above, at about 95% similarity with KT164088, you will begin to see a sprinkling of P. clavaeformis from East Tennessee.

At this point in the history of the FWGNA Blog, I have referred to my 2011 paper in Malacologia – the one that synonymized Goniobasis & Elimia under Pleurocera [8] – so often that I imagine you all are sick of it [9].  That was an allozyme paper, involving 9 populations of P. clavaeformis from East Tennessee, 4 control populations of P. simplex, and a pair of carinifera/vestita populations from North Georgia, more about which later.  The summer that paper was published, John Robinson and I sequenced single individual CO1 haplotypes from each of those same 15 populations, in connection with an oral presentation we were planning for the 2011 AMS meeting in Pittsburgh.  We uploaded those 15 sequences to GenBank promptly, although our results were not ultimately published until quite recently [10].  The gene tree looks like this: 
The point that John and I were making in our little note had to do with the hazards of DNA barcoding.  A researcher who naively sets 5% sequence similarity as a cut point for species recognition, for example, will imagine that our 15 populations comprised 10 species, lumping only the 6 P. clavaeformis bearing the modal CO1 haplotype at the top of the figure.  And even at 10% sequence similarity, a naïve researcher would recognize 8 species, where (at most) only 4 exist.

Now here’s a mental exercise for you.  Imagine Figure 3 above rotated 180 degrees.  It will actually fit almost like a lock-and-key into the top half of Whelan & Strong’s Figure 2, like this: 

The modal CO1 sequence that Dillon & Robinson obtained from 6 of our 9 P. clavaeformis populations in East Tennessee is approximately 95% similar to the modal “prasinata/pyrenellum” sequence that W&S obtained from North Alabama.  And in fact, one of our outlying East Tennessee sequences (JF837315) is approximately 95% similar to a bunch of the outlying North Alabama sequences.  The bottom line is that all six North Alabama Pleurocera populations are genetically indistinguishable from East Tennessee P. clavaeformis.

I first preached a series of sermons on this topic back in 2009 [11], but The Spirit has moved me into the pulpit again.  The evolutionary relationships between the malacofaunas of the Tennessee River and the Mobile Basin are much closer than anyone has ever realized.  Nineteenth-century taxonomic tradition has always held that the two pleurocerid faunas are completely disjoint, sharing no species whatsoever.  But the shell morphological intergradation we see in East Tennessee from acutocarinata to clavaeformis to unciale to curta is strikingly parallel to the intergradation from carinifiera to vestita to praesinata to foremani observed in the Mobile Basin.  That was my rationale for analyzing a carinifera/vestita set from North Georgia together with the three sets of acutocarinata/clavaeformis/unciale I sampled from East Tennessee in 2011.  It seemed possible to me that all 11 populations might prove conspecific.

On the basis of the ten polymorphic allozyme-encoding loci I analyzed in 2011, the genetic relationship between the two North Georgia populations and the nine East Tennessee populations was ambiguous.  As were the CO1 results I subsequently obtained with John Robinson, reproduced above.  Ultimately I decided to refer to the entire basket-full of Tennessee/Mobile Basin populations sharing this similar, slippery shell morphology as the “carinifera group,” and set the matter aside [12, 13].

The results of Whelan & Strong have reawakened the issue, and brought it roaring back to the fore.  In North Alabama, apparently Pleurocera “prasinata” of the Mobile Basin is genetically and morphologically indistinguishable from Pleurocera clavaeformis of the Tennessee.  This is big news [14].

Science is the construction of testable hypotheses about the natural world.  So I will concluded my long, rambling series of essays on the North Alabama Pleuroceridae with some science.  Pleurocera prasinata populations are moderately common in the main Coosa River and in scattered larger tributaries downstream from the North Georgia populations of P. carinifera and P. vestita I sampled for my 2011 paper [15].  I hypothesize that Coosa River populations of P. prasinata are more genetically similar to North Georgia carinifera and vestita than they are to the Cahaba populations of P. prasinata sampled by Whelan & Strong.  I challenge any of my colleagues with research interests in the Alabama Pleuroceridae to test my hypothesis, using any genetic tools at their disposal.  We’ll be standing by.


Notes:

[1] Previous installments in this series:
  • Mitochondrial Superheterogeneity: What we know [15Mar16]
  • Mitochondrial Superheterogeneity: What it means [6Apr16]
  • Mitochondrial Superheterogeneity and Speciation [3May16]
  • The Shape-shifting Pleurocera of North Alabama [2June16]
[2] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  Open Access: [html]

[3] Dillon, R.T., and C. Lydeard (1998) Divergence among Mobile Basin populations of the pleurocerid snail genus, Leptoxis, estimated by allozyme electrophoresis.  Malacologia. 39: 111-119. [PDF]

[4] The estimated 2% sequence divergence values shown in Figure 1 were obtained by blasting a randomly chosen modal (clade 6) pyrenella CO1 sequence, KT164127, against randomly-chosen Clade 7 sequence KT164125 and Clade 8 sequence KT164088.

[5] The estimate of 8% mtDNA sequence shown in Fig 2 above was obtained by blasting modal (clade 6) CO1 sequence KT164127 against clade 3 CO1 sequence KT164138.

[6] The first-most amazing thing is CO1 sequence KT163940, that single blue dot showing at Red-star-14 in the final figure of my May post.

[7] Well, to be precise, one individual was excluded by misidentification, and no CO1 sequence was obtained for 6 others.  So 113 North Alabama Pleurocera CO1 sequences, to be precise.

[8] Dillon, R. T. (2011)  Robust shell phenotype is a local response to stream size in the genus Pleurocera (Rafinesque 1818).  Malacologia 53: 265-277. [PDF]

[9] Goodbye Goniobasis, Farewell Elimia [23Mar11]

[10] Dillon, R. T. Jr, and J. D. Robinson (2016)  The hazards of DNA barcoding, as illustrated by the pleurocerid gastropods of East Tennessee.  Ellipsaria 18(1): 22-24. [PDF]

[11] My 2009 series on genetic relationships in the Mobile Basin Pleuroceridae:
  • Mobile Basin I: Two pleurocerids proposed for listing [24Aug09]
  • Mobile Basin II: Leptoxis lessons [15Sept09]
  • Mobile Basin III: Pleurocera puzzles [12Oct09]
  • Mobile Basin IV: Goniobasis WTFs [13Nov09]
[12] As I pointed out in both my essay of 12Oct09 and in my 2011 paper, the oldest name in the basket seems to be Lamarck’s (1822) Melania carinifera.  Lamarck gave his locality as “pays des Chérokées, dansun ruisseau qui se jette dans la rivière d'Estan-Alley,” but I don’t think any English speaker in 200 years has ever had a clue where “la rivière d'Estan-Alley” might be.  Binney (1864) simply abbreviated Lamarck’s locality as “Cherokee Country,” which Tryon (1873) understood to mean Cherokee County, Georgia.  Cherokee county lies in North Georgia’s Etowah River valley, draining SW toward the Alabama/Coosa and the Mobile Basin.  So both Goodrich (1941) and Thompson (2000) followed Tryon in restricting carinifera to tributaries of the Mobile Basin, and I don’t see any reason to question that call here in 2016.  I don’t think any pleurocerid populations looking like Lamarck’s carinifera actually inhabit Cherokee County today, but that whole region has been impacted by the Atlanta sprawl.  Pleurocera populations matching Lamarck’s carinifera do indeed inhabit Etowah tributaries in other North Georgia counties nearby, such as Whitfield, from whence my 2011 population was sampled.

[13] So following the logic above, I seriously considered entitling the present essay “Pleurocera carinifera in the Tennessee Basin,” not “Pleurocera clavaeformis in the Mobile Basin.”  But ultimately I decided that this entire topic is already sufficiently complex and obscure.  There aren’t five people in the world who know what I mean when I say “Pleurocera clavaeformis,” and if I swapped over to Pleurocera carinifera, I’d confuse even those.

[14] Here’s another mental exercise.  In what sense of the adjective “big” is this news big?

[15] Here I’m looking at a 1993 gray literature report submitted to the Alabama Natural History Program by Art Bogan and Malcolm Pierson entitled, “Survey of the Aquatic Gastropods of the Coosa River Basin, Alabama: 1992.”

Thursday, June 2, 2016

The Shape-shifting Pleurocera of North Alabama


The Tennessee River south of Chattanooga reminds me of Danica Patrick on three tires.  Swerving wildly out of the fertile Valley & Ridge Province of East Tennessee, she slices through the Cumberland Plateau at Walden Ridge, bounces off the NW Georgia guard rail, and plunges into the Interior Plains of North Alabama.  The character of the freshwater gastropod fauna changes slightly but perceptibly – not so much in the main river as in the tributaries.  The little black Pleurocera simplex populations, so common in springs and small streams of East Tennessee, disappear west of Chattanooga, to be replaced by populations of the larger and more impressively-shelled P. laqueata.

The most conspicuous element of the Tennessee River macrobenthos through this entire region is Pleurocera canaliculata (Say, 1821) bearing shells of the typical, robust form.  The smaller tributaries are inhabited by a more slender and lightly-shelled ecophenotypic variant, originally described by T. A. Conrad in 1834 as “Melania pyrenellum.”  In 2013 Stephen Jacquemin, Mark Pyron and I presented evidence that the distinction between Conrad’s pyrenellum in small tributaries and Say’s canaliculata in the main Tennessee River is “cryptic phenotypic plasticity,” intrapopulation morphological variance so extreme as to prompt an (erroneous) hypothesis of speciation [1]

Fig 1. Typical Pleurocera canaliculata from the Tennessee R at Site #1.
The sample of typical P. canaliculata canaliculata depicted in Figure 1 above was collected from the Tennessee River at Decatur, Alabama – marked as site #1 on the map below [2].  Stephen, Mark, and I showed that this population is conspecific with the population of P.canaliculata pyrenellum depicted in Figure 2 below, collected from Limestone Creek at Site #2.  I reviewed the evolutionary biology of P. canaliculata throughout eastern North America generally in a pair of related essays posted on this blog back in June of 2013 [3].

Fig 2.  Pleurocera canaliculata pyrenellum from Limestone Creek at Site #2.
The situation is similar in the case of P. clavaeformis, another notorious shape-shifter.  Pleurocera clavaeformis is the most common freshwater gastropod in East Tennessee, ranging from small creeks into large rivers, although not inhabiting the main Tennessee River itself.  Populations of clavaeformis bearing more robust shells in the larger rivers were for many years identified (erroneously) as the distinct species Pleurocera unciale and P. curtum [4].  I first documented cryptic phenotypic plasticity in East Tennessee populations of P. clavaeformis in February of 2007, returning to the subject again in October 2009 and March 2011 [5].

Fig 3.  The Tennessee River west of Knoxville.
Populations of P. clavaeformis are common in several of the Tennessee tributaries of North Alabama, including the Elk, Flint, and Paint Rock Rivers.  These populations generally seem to bear robust shells, however, and have historically been identified as “Pleurocera curtum.”  Goodrich, in fact, stated that Pleurocera (Goniobasis) clavaeformis “does not make the westward turn around Walden Ridge” because he was unaware of any North Alabama populations bearing shells of the lighter, more typical form inhabiting streams of the Cumberland Plateau or Interior Plains [6].

I myself cannot boast of extensive field experience in the state of Alabama.  But I collected the mixed sample of P. clavaeformis and P. canaliculata shown in Figure 4 below from the Paint Rock River SE of Huntsville in March of 1988 (marked as Site #4 above).  The shell variation was rather dramatic in the P. clavaeformis population (top row), ranging from medium-typical to robust-curtum.  The P. canaliculata population (bottom row) was more uniformly heavy-shelled, ranging only from skinnyish-typical to plain-vanilla-typical.

Fig 4.  Pleurocera clavaeformis and P. canaliculata from the Paint Rock at Site 4.
Most significantly, notice that my 1988 sample of Pleurocera from the Paint Rock River did not include any shells demonstrating the pyrenellum morphology, as depicted in Figure 2.  Phenotypic plasticity manifests itself most cryptically where small streams like Limestone Creek empty directly into large rivers, like the Tennessee.  In such situations, the population bearing the more gracile shell morphology will differ strikingly from the population bearing the robust shell, sometimes even mixing unconformably at the mouth of the creek, looking like reproductively-isolated biological species.  But in rivers that grow larger gradually, such as the Paint Rock, the shell morphology of the pleurocerid population changes gradually, and taxonomists are usually not fooled.

It is perhaps for this reason that Goodrich [6] did not report Pleurocera of the pyrenellum form from the Paint Rock River drainage, or indeed from any of the larger tributaries of the Tennessee in North Alabama, such as the Flint or the Elk.  Goodrich gave the distribution of pyrenellum as “tributaries of the Tennessee River in Morgan and Limestone Counties” downstream from the Paint Rock, “and Walker County, Georgia” upstream from the Paint Rock, but not in Madison or Jackson Counties, through which the Paint Rock River runs.  The canaliculata population of the Paint Rock subdrainage just looks like typical canaliculata.  It doesn’t fool anybody.

So for the last three months, we’ve been studying (in excruciating detail) the recent paper by Nathan Whelan and Ellen Strong on mtDNA sequence divergence among the pleurocerid populations of North Alabama [7].  Previously our attention has focused on populations of Leptoxis [8].  But Whelan & Strong also sampled five populations they identified as “Pleurocera prasinata” from tributaries of the Cahaba River draining south through Alabama into the Mobile Basin, and one population they identified as “Pleurocera pyrenellum” from a tributary of the Paint Rock River in Jackson County [9].  That Paint Rock tributary site is marked as Site A on the map above.

Whelan & Strong's Fig 7.  Row C shows putative "Pleurocera pyrenella."  
Whelan & Strong’s Figure 7 is reproduced above, showing example shells from their sample of “Pleurocera pyrenellum” on Row C, at the bottom.  These specimens do not look like the pyrenellum form of P. canaliculata to me.  The shells borne by pyrenellum are flat-sided, almost trapezoidal, essentially demonstrating no suture or indeed any whorl, as shown in Figure 2.  North Alabama pyrenellum populations also typically bear shells marked with spiral cords, especially around the aperture.  But the shells depicted in Row C of Whelan & Strong’s figure seem to lack spiral cords, and demonstrate the slightly rounded whorls and impressed sutures typical of Pleurocera clavaeformis

I hasten to stipulate that I have no personal observations from the Paint Rock subdrainage as far upstream as the Whelan & Strong sample.  And of course, the entire theme of the present essay has been one of caution regarding the use of shell morphology to distinguish species of pleurocerids.  So what might the mtDNA sequence data tell us about the genetic relationships between Whelan & Strong’s Pleurocera populations (all six of them) and Pleurocera populations sampled from elsewhere in the drainage of the Tennessee?  Tune in next time…


Notes:

[1] Dillon, R. T., S. J. Jacquemin & M. Pyron (2013)  Cryptic phenotypic plasticity in populations of the freshwater prosobranch snail, Pleurocera canaliculata.  Hydrobiologia 709: 117-127.  [PDF]

[2] Here are the complete locality data for all four sites mentioned in this month’s blog:
  • Site #1 – Tennessee River at Decatur, Morgan County, AL.  (34.6279N; -86.9564W).  This was site “CT” of Dillon et al. (2013).
  • Site #2 – Limestone Creek at Nick Davis Road, 2 km N of Capshaw, Limestone County, AL. 
  • (34.8027N; -86.8163W).  This was site “PT” of Dillon et al. (2013).
  • Site #4 - Paint Rock River at US 431 bridge, 25 km SE of Huntsville, AL.  (34.4992, -86.3905).
  • Site A – Estill Fork at Co. 140 bridge, Jackson Co, AL.  (34.9653, -86.1537).
[3]  Cryptic phenotypic plasticity in P. canaliculata:
  • Pleurocera acuta is Pleurocera canaliculata [3June13]
  • Pleurocera canaliculata and the process of scientific discovery [18June13]
[4] Dillon, R. T. (2011)  Robust shell phenotype is a local response to stream size in the genus Pleurocera (Rafinesque 1818). Malacologia 53: 265-277. [PDF]

[5] Cryptic phenotypic plasticity in P. clavaeformis:
  • Goodrichian Taxon Shift [20Feb07]
  • Mobile Basin III: Pleurocera Puzzles [12Oct09]
  • Goodbye Goniobasis, Farewell Elimia [23Mar11]
[6] Goodrich, C. (1940) The Pleuroceridae of the Ohio River system. Occas. Pprs. Mus. Zool. Univ. Mich., 417, 1-21.

[7] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  Open Access: [html]

[8]  I have featured the Leptoxis mtDNA data set of Whelan & Strong in three previous essays:
  • Mitochondrial Superheterogeneity: What we know [15Mar16]
  • Mitochondrial Superheterogeneity: What it means [6Apr16]
  • Mitochondrial Superheterogeneity and speciation [3May16]
[9] Our good friends Nathan Whelan and Ellen Strong seemed unaware that Conrad’s pyrenellum is an ecophenotypic variant of Say’s canaliculata, and indeed entirely neglected to cite the (2013) paper by Jacquemin, Pyron, and myself.  I’m sure this was just a simple oversight.

Tuesday, May 3, 2016

Mitochondrial Superheterogeneity and Speciation


Editor’s Note – This is the third essay in my series on the phenomenon of double digit intrapopulation mtDNA sequence variation, which I have begun calling “mitochondrial superheterogeneity.”  The points I plan to advance here will depend upon my crazy “wildebison” model of 6Apr16, which (in turn) depended on the field observations I reviewed in my essay of 15Mar16.

Understood in their proper context, typical mtDNA sequence data sets yield weak, null models of population relationship, not directly relevant to the question of speciation [1], but certainly correlative.  A mitochondrial gene sequence is a perfectly fine single character.  Given a decent sample size per population, typical mtDNA sequence data can reasonably be used to estimate interpopulation genetic divergence, which (especially in combination with data from other sources) is generally correlated with the likelihood of speciation [2].

So in the last couple months we have focused on the mtDNA sequence data collected by Whelan & Strong from five populations of Leptoxisampla” inhabiting tributaries of the Cahaba River, draining south through Alabama into the Mobile Bay [3].  Whelan & Strong also sampled n = 20 Leptoxis praerosa from a tributary of the Tennessee River, draining west across the top of Alabama toward the Mississippi River.  Their data set included many striking examples of mitochondrial superheterogeneity.  So while intriguing as an evolutionary phenomenon, doesn’t the occurrence of up to 20% sequence divergence within conspecific populations of “ampla” compromise the utility of mtDNA as a tool for distinguishing “ampla” from praerosa?

No, perhaps just the opposite.  But before I develop that surprising point, let’s back up a few years and review what we actually know about the evolutionary biology of Leptoxis populations in Alabama.

The Leptoxis fauna of the Mobile Basin was last comprehensively monographed by Calvin Goodrich in 1922 [4], prior to his 1934 - 1941 awakening to the Modern Synthesis [5].  The entire pleurocerid fauna of Alabama was then decimated by a massive program of impoundment and channelization, leaving behind what may be the single biggest taxonomic mess anywhere in North American malacology [6].

In 1998, Chuck Lydeard and I published a survey of genetic variation at 9 allozyme-encoding loci in eight populations representing all four nominal species of Leptoxis known (at that time) to persist in the Mobile Basin of Alabama [7], calibrated with three populations of the widespread and well-studied Leptoxis praerosa of Tennessee.  Our Alabama populations included three identified as Leptoxis ampla from the Cahaba drainage [8], two populations identified as L. taeniata from tributaries of the Coosa River, and one population of Leptoxis picta from the main Alabama River way downstream in Monroe County [9].  The allozyme variation among those six populations was comparable to that observed among our three control populations of Leptoxis praerosa, and negligible compared to the genetic divergence between praerosa and the ampla+taeniata+picta group combined.  Thus our (9 gene, 11 population, 333 individual) results suggested that Leptoxis ampla (Anthony 1855) is a junior synonym of L. picta (Conrad 1834) [10].


So returning to the mtDNA sequence data of Whelan & Strong.  It is reassuring to note that, despite the high incidence of superheterogeneity W&S observed scattered among the five Cahaba drainage pleurocerid populations of (what should better be referred to as) Leptoxis picta, their 16S+CO1 gene tree nevertheless depicted the single population of Leptoxis praerosa they sampled from that Tennessee tributary as genetically distinct.

The bottom half [11] of Whelan & Strong’s Figure 2 (reproduced above) shows negligible sequence divergence within their 20-individual sample of L. praerosa, but approximately 13% divergence between modal ("clade 9") L. praerosa and modal (“clade 15”) L. picta [12].  Granted, the sequence variance within populations of L. picta (“ampla”) does indeed range well above 13%.  But a mode-to-mode difference of such magnitude is certainly consistent with an hypothesis of speciation.

And we have thus far given short shrift to the histone H3 sequence data adduced by Whelan & Strong, which is especially interesting because the gene is nuclear, and tends to evolve quite slowly.  Figure 3 of W&S (reproduced below) shows essentially zero sequence divergence within or among any of the five L. picta (“ampla”) populations, but 2.0% divergence between picta and praerosa [13].  



Levels of genetic divergence in allopatry, no matter how striking, do not directly address the question reproductive isolation, and hence can only constitute indirect evidence of speciation.  But the nine allozyme-encoding genes of Dillon & Lydeard, plus the 13% mode-to-mode sequence divergence W&S report for the CO1 gene, plus the 2.0% sequence difference at histone H3, taken together with whatever morphological evidence may have accumulated [14], certainly combine to suggest that the specific distinction between L. picta (aka “ampla”) and L. praerosa is indeed a valid one.

Now begging your indulgence, let me return to Figure 4 of Whelan & Strong – the third time I have reproduced the figure below in three months.  And let’s focus once again on the situation at red-star-14 in the lower left corner, showing that the Shades Creek Leptoxis sample included 16 copies of the modal haplotype and 1 copy of a 12% divergent haplotype, modal in the Cahaba River at US52.  (The red-star numbers in Fig 4 below correspond to the clade numbers in Fig 2 above.)  Last month I suggested that cases of mitochondrial superheterogeneity such as this are the signatures of very rare dispersal events, as though a wildebeest were airlifted into a herd of bison, and successfully interbred.  Seen in this light, the results depicted at red-star-14 constitute direct, positive evidence that the Leptoxis population of Shades Creek is conspecific with the Leptoxis population of Cahaba-US52.


And similarly at red-star-12.  Here we see that the Leptoxis populations of Cahaba-Bibb/Shelby and Cahaba-US82 share a unique, rare CO1 haplotype that Whelan & Strong didn’t discover modally anywhere.  This would seem to constitute direct, positive evidence that the Bibb/Shelby and the US82 Leptoxis populations are conspecific both with some third (unsampled) population where that particular haplotype is much more common, and with each other, as well.

So more broadly, I would suggest that rare, superdivergent mtDNA haplotypes be understood as markers of genetic compatibility with second populations some distance removed.  And at red stars 10, 11, and 13, we see three additional markers of genetic compatibility with three other populations of Leptoxis that Whelan & Strong did not sample.  All of these populations must be conspecific – the populations of Leptoxis picta in which W&S found the markers, and the four other populations elsewhere.  In an ideal world, given complete sampling, it seems possible to me that the phenomenon of mitochondrial superheterogeneity might afford a direct, positive tool to reconstruct specific relationships among extensive sets of highly allopatric populations in their entirety.

Well, perhaps you’d like to join us back here in the real world, Dillon?  The pleurocerid fauna of real Alabama is just a ghost of its former self.  And the Leptoxis populations where the sequences shown at red stars 10, 11, 12 and 13 are modal may well have been extinguished 80 years ago.  Or longer?  And those mtDNA haplotypes might be genetic fossils, of populations long gone to glory?  Interesting to think about.

But turning the page.  As I am sure most of my readership will have by now noticed, the mtDNA survey of Whelan & Strong extended to cover six populations of Pleurocera, sampled alongside the six populations of Leptoxis upon which we have focused these last three months.  Their Pleurocera results offer some striking contrasts with their Leptoxis results, one of which is illustrated in the copy of their Figure 3 I have reproduced above.  Why doesn’t the population W&S identified as “Pleurocera pyrenella” from the Tennessee drainage appear genetically distinct from the five populations they identified as “Pleurocera prasinata” from the Mobile Basin?  Tune in next time!


Notes

[1]  Gene trees and species trees are not the same thing:
[2] Examples of the correlative relationship between mtDNA sequence divergence and speciation:
  • Rhodacmea Ridotto [8Aug11]
  • The Lymnaeidae 2012: Stagnalis yardstick [4June12]
[3] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  Open Access: [html]

[4] Goodrich, C. (1922) The Anculosae of the Alabama River Drainage. Misc. Publ. Univ. Mich Mus. Zool. 7: 1-57.

[5] Goodrich’s (1934 – 1941) “Studies of the Gastropod Family Pleuroceridae” series may represent the earliest application of the modern evolutionary synthesis to malacology – I don’t know of any earlier.  For more, see my essays:
  • The Legacy of Calvin Goodrich [23Jan07]
  • Mobile Basin II: Leptoxis lessons [15Sept09]
[6]  But we can’t blame the entire mess on Alabama Power Company and the Corps of Engineers.  As was true throughout The South, the state of Alabama was blanketed with cotton and other row crop agriculture through the nineteenth century and well into the twentieth.  Bank-to-bank farming dumped tremendous loads of silt into the smaller rivers and streams, which still chokes most of the pleurocerid habitat, even to this day.

[7] Dillon, R.T., and C. Lydeard (1998) Divergence among Mobile Basin populations of the pleurocerid snail genus, Leptoxis, estimated by allozyme electrophoresis. Malacologia 39: 111-119. [PDF]

[8] One of the three Leptoxis ampla sites of Dillon & Lydeard matched one of the five Whelan & Strong sites precisely – the Cahaba River at HWY 52 bridge.

[9] We also included two populations of Leptoxis plicata from the Black Warrior drainage, not relevant to this particular essay.  We did not include the subsequently rediscovered Leptoxis “foremani/downiei” or Leptoxis “compacta.”

[10]  Our good friends Nathan Whelan and Ellen Strong neglected to cite Dillon & Lydeard (1998) in their more recent work on the Alabama Leptoxis under review here.  I’m sure this was just a simple oversight.

[11] The upper half of Whelan & Strong’s Figure 2 depicted the 16S+CO1 genetic relationships among their six populations of Pleurocera.  We’ll return to those data next month.

[12] For this 13% estimate I blasted randomly-chosen L. praerosa CO1 sequence KT164014 against randomly-chosen clade 15 L. picta sequence KT163938.

[13] To obtain this 2.0% estimate, I blasted randomly-chosen L. praerosa H3 sequence KT164460 against randomly-chosen L. picta (“ampla”) H3 sequence KT164387, just as in note [12] above.

[14]  Whelan & Strong did not offer any morphological observations on L. praerosa.  But it is my subjective impression that there may be subtle differences in the whorl shape or spire height between praerosa and picta (“ampla”) – perhaps noticeable only in juveniles, or in adults inhabiting calmer, more protected waters [15].

[15] And it is a crying shame that the captive-rearing study published by Whelan and colleagues (2015) in J. Moll. Stud. 81:85-95 was not controlled for current speed.

Wednesday, April 6, 2016

Mitochondrial Superheterogeneity: What it means


Last month [1] we reviewed, or at least touched upon, 15 separate reports of double-digit mtDNA heterogeneity within gastropod populations published over the last 20 years, the paper by Whelan & Strong [2] among the best and most recent.  Three additional reports from pulmonate snail populations have subsequently come to my attention [3].  The discussion sections of almost all of these works have featured lists of possible explanations for the phenomenon, including population fragmentation, great antiquity, introgression from other species and cryptic speciation, many of which do not seem to fit the data as it has now accumulated, none of which is complete. 

So Rob Dillon’s model to explain mitochondrial superheterogeneity begins with an observation (not an assumption, in the case of pleurocerid snails) of large numbers of super-isolated populations.  Then I add two more super-assumptions: super-long time and super-rare dispersal events.  And let me begin with an extended analogy.

The even-toed ungulates first evolved in the Eocene Epoch, about 50 mybp.  So today, the mtCOI sequence divergence between American bison (JF443195) and the wildebeest of the Serengeti (JX436977) is approximately 15%.  I suggest that when we stand on the bank of the Green River in Polk County, NC, and look down at the population of Pleurocera proxima grazing over the rocks, it is as though we were Lewis and Clark, looking over a vast herd of American bison [4].  And should we drive 80 km west to Buncombe Co, NC, and look down on the Pleurocera proxima population inhabiting Bent Creek, it is as though we were Frederick Russell Burnham standing before the wildebeests. 

Now suppose that every million generations or so, a wading bird with sticky feet transports one snail from Buncombe County to Polk County, as though a modern zookeeper were to airlift a wildebeest to Wyoming.   I don’t know, but it seems likely to me that our jet lagged wildebeest might find itself perfectly well-adapted to graze on the plains of Wyoming in all respects, morphologically and physiologically. 

But it does not seem likely to me that a single wildebeest could reproduce in a herd of American bison.  I imagine a variety of prezygotic reproductive isolating mechanisms have evolved between wildebeest and bison – appearances, smells, behaviors and so forth.  And even if a mating should occur, I feel certain all sorts of postzygotic incompatibilities have evolved - the chromosomes of the two species probably don’t match, a million screw ups occur in development, and nothing comes of it.

So (I admit) the most implausible element of my model is that, in the case of pleurocerid snails, a super-rare immigrant arriving via sticky-bird express must successfully mate within a host population from which it has been isolated for millions of generations.  There are several factors that make this phenomenon marginally less-implausible for pleurocerids, specifically.  Pleurocerid snails are aphallic, and no obvious mating behavior has been reported.  Apparently the male simply crawls over the female and deposits a spermatophore in her gonoduct, so unceremoniously that human observers typically don’t even notice it.  There’s very little light down where the snails live, so appearances don’t matter.  And pleurocerid populations typically inhabit waters with significant flow rates, so it seems unlikely that chemical mating cues could evolve.

Now why postzygotic reproductive incompatibilities have not evolved over millions of generations among such isolated gastropod populations, I do not know.  North American pleurocerids seem to demonstrate negligible anatomical diversity [5], and negligible chromosomal diversity to match [6].  I was tempted to add “super-stable environment” to my list of assumptions in paragraph two at the top of this essay, because it seems possible to me that stabilizing selection may have worked to prevent reproductive isolating mechanisms from evolving.

But I should hasten to add that very little of the reproductive biology I have reviewed directly above applies to pulmonate land snails, and that it was in land snail populations mitochondrial superheterogeneity was first discovered [7].  The bottom line seems to be that, over a wide range of mating systems, populations as isolated as bison and wildebeest have not speciated in gastropod world.  The plains of snail-America and snail-Africa are both grazed by reproductively compatible “wildebison.” 

Rob Dillon’s patented “wildebison model” fits all six of the points I reviewed last month regarding mitochondrial superheterogeneity as a general phenomenon [1].  So let us return to the Leptoxis population inhabiting Shades Creek in northern Alabama, marked in dark blue above.  Whelan & Strong reported 16 copies of the modal CO1 sequence (the “bison” sequence, at red-star-15) and one copy of a 12% divergent sequence, which we’ll call “wildebeest.”  The wildebeest sequence almost exactly matched the modal sequence in the Cahaba River at the US52 bridge, shown in black above (at red-star-14).  And not only did the rare CO1 sequence of Shades Creek match the modal CO1 sequence of the Cahaba River, the rare 16S sequence of Shades Creek matched the modal 16S sequence of the Cahaba River.  The conclusion seems almost inescapable that at some point in the (probably distant) past, a Cahaba wildebeest has been airlifted into the Shades Creek bison population.

The first-most amazing thing about this data set is that an individual Cahaba wildebeest was apparently able to mate into the Shades Creek bison herd, despite millions of generations of isolation.  And the second-most amazing thing is that Whelan & Strong happened to sample both Shades Creek and the Cahaba River, to show us what happened.  In most data sets of this sort, we simply find no clue. 

So for example, in addition to the 17 copies of the modal CO1 sequence Whelan & Strong reported from the Cahaba River at US52, they also reported two copies of a 20% divergent sequence that matches nothing else in their database, way off at red-star-11 [8, 9].  Did that rare mitochondrial genome come from a third population of “wildecamels,” currently grazing on some other continent that W&S did not sample?  Or might that genome have originated in an extinct population of “wilde-Irish-elk,” now gone?   A fascinating question.

I will close with two final notes.  First, I should emphasize that I have not evoked selection at any point during the essay above.  I very nearly used the S-word when I was waving my hands about the apparent absence of reproductive isolation among highly isolated gastropod populations with a surprising range of evolutionary backgrounds, but (ultimately) held off.  The wildebison model is grudgingly neutral.

And second, my good friend John Robinson and I are currently simulating the evolution of mitochondrial superheterogeneity using the “ms” program of R. R. Hudson [10]. Our preliminary results have not yielded the phenomenon in single-population models given any reasonable values of mutation rate (u) and population size (Ne).  Our two-population models do, however, yield mitochondrial superheterogeneity within reasonable values of u and (perhaps-surprisingly low values of) Ne, given (super-rare) migration rates around Nem = 0.0001 genomes per generation.

I find all this quite interesting as a study of evolutionary science, pure as the driven snow, and (I feel sure) a large fraction of my readership does as well.  But in situations such as the pleurocerid fauna of Alabama, research efforts have been at least partly motivated by conservation concerns regarding the distribution of potentially endangered species.  What might mtDNA sequence data sets such as developed by Whelan & Strong, riddled (as they are) with superheterogeneity, tell us about the species relationships of the populations sampled?  Tune in next time!


Notes

[1] Mitochondrial Superheterogeneity: What we know [15Mar16]

[2] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  Open Access: [html]

[3] Pinceel, J, K. Jordaens & T. Backeljau (2005)  Extreme mtDNA divergences in a terrestrial slug (Gastropoda, Pulmonata, Arionidae): Accelerated evolution, allopatric divergence and secondary contact.  J. Evol. Biol. 18: 1264 – 1280.  Parmakelis, A., P. Kotsakoizi & D. Rand (2013)  Animal mitochondria, positive selection and cyto-nuclear coevolution: Insights from Pulmonates.  PLOS ONE 8(4): e61970.  Nolan, J. R., U. Bergthorsson & C. M. Adema (2014)  Physella acuta: atypical mitochondrial gene order among panpulmonates (Gastropoda).  J. Moll. Stud. 80: 388 – 399. 

[4] The populations of Pleurocera I’m using as examples here were sampled in Dillon, R T. and J. D. Robinson (2009)  The snails the dinosaurs saw: Are the pleurocerid populations of the Older Appalachians a relict of the Paleozoic Era?  Journal of the North American Benthological Society 28: 1 - 11. [PDF].  I reviewed the Dillon & Robinson results in my blog post of March, 2009.  See:
  • The Snails The Dinosaurs Saw [16Mar09]
[5] Dazo B (1965) The morphology and natural history of Pleurocera acuta and Goniobasis livescens (Gastropoda: Cerithiacea: Pleuroceridae). Malacologia 3:1–80.   Strong, E., 2005. A morphological reanalysis of Pleurocera acuta Rafinesque, 1831, and Elimia livescens (Menke, 1830) (Gastropoda: Cerithioidea: Pleuroceridae). Nautilus 119: 119–132

[6] Dillon, R.T. (1989) Karyotypic evolution in pleurocerid snails. I. Genomic DNA estimated by flow cytometry. Malacologia 31: 197-203. [PDF]   Dillon, R.T. (1991) Karyotypic evolution in pleurocerid snails. II. Pleurocera, Goniobasis, and Juga. Malacologia 33: 339-344.  [PDF

[7]  I almost cut the two paragraphs about the absence reproductive isolating mechanisms in pleurocerid snails entirely out of this essay.  But then I figured, hell, if the land snail people don’t like it, they can start their own blog.

[8]  For this calculation I used KT164003 as an example as the modal CO1 sequence from the Cahaba River at US52, and KT164004 as an example of the rare, divergent sequence, and compared the two using blastn.

[9] And somewhat amazingly, Whelan & Strong also found one mitochondrial haplotype in the Cahaba River at US52 that matches the Shades Creek mode.  So their Figure 4 (reproduced above) should have shown one little black circle near their big blue circle, as well as that single little blue circle near their big black.  I have added one little black circle at red-star-15 above.  More about this next month.

[10] Hudson, R. R. (2002)  Generating samples under a Wright-Fisher neutral model of genetic variation.  Bioinformatics 18: 337-338.  

Tuesday, March 15, 2016

Mitochondrial Superheterogeneity: What we know


Finally some decent sample sizes, thank heaven!  I remember thinking this to myself when the abstract of the recent paper in Zoologica Scripta by Nathan Whelan and Ellen Strong fell upon my eyes in January [1].  Perhaps now, after twenty years of bouncing around on the fringes of evolutionary science, the phenomenon we here define as “mitochondrial superheterogeneity” will finally attract the attention it so richly deserves.

A population can be said to demonstrate “mitochondrial superheterogeneity” when two or more of its members demonstrate 10% sequence divergence or greater for any single-copy mtDNA gene, not sex-linked.  The phenomenon was first documented (as far as I am aware) in 1996 by Thomaz, Guiller and Clarke in European populations of the land snail, Cepaea nemoralis [2].  It has now been reported at least three additional times in land snails [2], twice in populations of freshwater pulmonates [3], once in oriental pomatiopsids [4], three times in Old World cerithiaceans [5] and six times in populations of North American pleurocerids [6, 7].  But the new study by Whelan & Strong is by far the most thorough.

W&S sampled 20 individuals from each of 12 populations of pleurocerid snails collected from North Alabama, sequencing three genes from all 240 individuals [8]: (mitochondrial) COI, (mitochondrial) 16S rRNA, and (nuclear) histone H3.  Six of the populations sampled were of Leptoxis (identified as praerosa in Tennessee drainages or ampla in Alabama drainages) and six of the populations were of Pleurocera (identified as pyrenella in Tennessee drainages or prasinata in Alabama drainages).  The specific identities of these populations are a complicated question, which we will defer to a later essay.  But here are the important findings:
(1) No intermediate sequences.  Given the small numbers of haploid genomes sampled in previous studies, it has seemed possible that individuals bearing mtDNA sequences differing by as much as 10% within populations might be united by (unsampled) individuals bearing intermediate sequences 2%, 4%, 6% and 8% divergent.  But apparently not. 

For example, the COI results obtained by W&S from five of their Leptoxis populations are depicted above.  The 19 snails [8] sequenced from the Shades Creek population (shown in dark blue) had three strikingly different haplotypes: N=16 in the modal category, N=2 about 68 mutational steps (9%) different from the modal, and N=1 about 88 steps (12%) different.  Notice that the red intermediate points in the figure below are mostly quite distant from the branch tips and entirely hypothetical.

(2)  Peculiar patterns of interpopulation haplotype sharing.  Let us focus on that single Shades Creek snail demonstrating a 12% COI sequence divergence from its N = 16 associates, marked with a red star in the figure above.  That CO1 sequence (KT163940) is only 1% different from the sequence demonstrated by N=17 of the 20 snails sampled from the Cahaba River at the US52 bridge, 5 km downstream and then back up the main river about 20 km (e.g., KT164013).

(3)  Tight coupling in the evolution and distribution of separate mitochondrial genes.  W&S confirmed that individuals bearing highly divergent COI sequences also tend to bear highly divergent 16S sequences.  So the starred individual in the COI network above also demonstrated a 16S sequence rather strikingly divergent from the modal Shade Creek 16S sequence.  And that divergent 16S sequence also very nearly matched the modal sequence recovered from the Cahaba River at the US52 bridge [9].

Such results were first reported in 2004 by Dillon & Frankis [5], working with Pleurocera proxima in southern Virginia [10].  Our 2004 mtDNA sample sizes were very small – just three individuals from three P. proxima populations, plus single individuals of P. catenaria dislocata and P. semicarinata.  But we did sequence both the COI and 16S genes, giving us a total of 11 sequences for each gene.  All three of our P. proxima from Naked Creek matched in both sequences, and all three of our P. proxima from Cripple Creek matched in both sequences.  But the individuals we sampled from Nicholas Creek demonstrated superheterogeneity: the CO1 sequence of the (N=2) mode and the (N=1) minority differing by 16.9%.  And that same N=1 minority snail also differed from the (N=2) modal 16S sequence in Nicholas Creek by 14.1%.

(4)  No evidence of cryptic speciation.  Although the sample sizes of mtDNA sequence data in all three of my previously-published works on this subject have been quite small, all three have focused on pleurocerid populations for which I have tested Hardy-Weinberg equilibrium at multiple polymorphic allozyme loci with sample sizes of at least N = 30, and often much larger.  These are not weak tests, and if cryptic species had been present, I have reason to think that I would have found them [11].  But I have found no evidence of non-random mating in any population from which I have reported mitochondrial superheterogeneity [12].

Whelan & Strong have reinforced this result with some classical (i.e., entirely qualitative) studies of shell and radular morphology, pallial oviduct anatomy, and head-foot coloration in their 12 pleurocerid populations from North Alabama.  Their morphological observations are nicely illustrated and described in quaint, Victorian style (e.g., “Outermost denticle on each side delicate, flimsy, variable in position”).  Although W&S do note some intrapopulation variation in some morphological traits, none seemed to correspond with mtDNA clade.

(5) No evidence of NUMTs.  In some circumstances mitochondrial genomes or portions of genomes have been discovered transposed into the nucleus, yielding “NUclear MiTochodrial DNA” which, being “free to evolve,” can diverge greatly from the bona fide mtDNA genome.  W&S used a next-generation technique to sequence the entire mitochondrial genome of a Leptoxis individual from which they had obtained highly-divergent COI and 16S sequences with their standard (Sanger) methods.  The next-generation sequences matched the Sanger sequences, counter to the NUMT hypothesis.

(6)  No evidence of DUI.  Doubly-uniparental inheritance is the surprising situation where two highly-divergent mtDNA genomes have evolved within a single species, one transmitted through eggs and the other transmitted through sperm.  The phenomenon has been documented in a great variety of bivalve groups, including the freshwater unionids, but is unknown in gastropods.  And indeed, W&S could find no systematic differences in the mtDNA genomes isolated from the male and female samples of pleurocerids they analyzed.

So that’s what we know about mitochondrial superheterogeneity as of March 2016, and what doesn’t cause it.  But what, then, might a reasonable explanation be?  Are data such as these rudely yelling something important to us, in some language we do not understand?  Tune in next time!


Notes

[1] Whelan, N.V. & E. E. Strong (2016)  Morphology, molecules and taxonomy: extreme incongruence in pleurocerids (Gastropoda, Cerithiodea, Pleuroceridae). Zoologica Scripta 45: 62 – 87.  Open Access: [html]

[2] Thomaz, D., Guiller, A. & Clarke, B. (1996) Extreme divergence of mitochondrial DNA within species of pulmonate land snails. Proceedings of the Royal Society of London B: Biological Sciences, 263, 363–368.  Goodacre, S. L. & C. M. Wade (2001)  Patterns of genetic variation in Pacific island land snails: the distribution of cytochrome b lineages among Society Island Partula.  Biol. J. Linn. Soc. 73: 131-138.  Guillier, A. et al. (2001)  Evolutionary history of the land snail, Helix aspersa in the western Mediterranean: preliminary results inferred from mitochondrial DNA sequences.  Molecular Ecology 10: 81-87.  Haase, M. et al. (2003) Mitochondrial differentiation in a polymorphic land snail: evidence for Pleistocene survival within the boundaries of permafrost.  J. Evol. Biol. 16: 415-428.

[3] My good friend and colleague Amy Wethington documented a striking case of mitochondrial superheterogeneity (both COI and 16S) in a Charleston population of Physa acuta in 2003.  We never could get a separate paper through the reviewers, but the data were published in her (2004) dissertation.  Andrea Walther and colleagues had better luck with the reviewers in her (2006) paper on Laevapex fuscus.  For more, see:
  • Phylogenetic Sporting and the Genus Laevapex [20July07]
[4] Wilke, T. et al. (2006)  Extreme mitochondrial sequence diversity in the intermediate schistosomiasis host Oncomelania hupensis robertsoni: another case of ancestral polymorphism?  Malacologia 48: 143-157.

[5]  Facon, B. et al. (2003)  A molecular phylogeography approach to biological invasions of the New World by parthenogenetic thiarid snails.  Molecular Ecology 12: 3027-3039.  Lee, T., H. C. Hong, J. J. Kim & D. O’Foighil (2007)  Phylogenetic and taxonomic incongruence involving nuclear and mitochondrial markers in Korean populations of the freshwater snail genus Semisulcospira.  Molecular Phylogenetics and Evolution 43: 386-397.  Miura, O., F. Kohler, T. Lee, J. Li, & D. O’Foighil (2013)  Rare, divergent Korean Semisulcospira spp. mitochondrial haplotypes have Japanese sister lineages.  J. Moll. Stud. 79: 86 – 89.
I reviewed the (2007) paper by Taehwan Lee and his colleagues on this blog in February, 2008:
  • Semisulcospira II: A second message from The East [1Feb08]
[6] Dillon, R. T., and R. C. Frankis. (2004)  High levels of DNA sequence divergence in isolated populations of the freshwater snail, Goniobasis.  American Malacological Bulletin 19: 69 - 77.  [PDF].  Sides, J. D.  (2005) The systematics of freshwater snails of the genus Pleurocera from the Mobile River Basin. Ph.D. dissertation, University of Alabama.  Lee, T., J. J. Kim, H. C. Hong, J. B. Burch, and D. O’Foighil (2006)  Crossing the contintental divide: the Columbia drainages species Juga hemphilli is a cryptic member of the eastern North American genus Elimia.  J. Moll. Stud. 72: 314-317.  Dillon, R T. and J. D. Robinson (2009)  The snails the dinosaurs saw: Are the pleurocerid populations of the Older Appalachians a relict of the Paleozoic Era?  Journal of the North American Benthological Society 28: 1 - 11. [PDF]
I reviewed the Dillon & Robinson (2009) results in my blog post of March, 2009.  See:
  • The Snails The Dinosaurs Saw [16Mar09]
[7] The note of Dillon & Robinson (2016) is a special case.  John Robinson and I first reported mitochondrial superheterogeneity in East Tennessee populations of Pleurocera clavaeformis and P. simplex in an oral presentation at the 2011 meeting of the American Malacological Society in Pittsburgh.  We uploaded our COI sequences to GenBank shortly thereafter, and drafted a manuscript entitled, “When cometh our reformation?  Molecular typology meets population genetics in the pleurocerid gastropod fauna of east Tennessee,” which we were ultimately unable to publish.  Our 2011 results have just this month become available as a note in Ellipsaria:
Dillon, R. T. Jr, and J. D. Robinson (2016)  The hazards of DNA barcoding, as illustrated by the pleurocerid gastropods of East Tennessee.  Ellipsaria 18: 22-24. [PDF]
More about this little work in a future post.

[8] Well, to be precise, one of the 240 snails was excluded by misidentification.  Then no COI sequence was ultimately obtained for 13 individuals, no 16S sequence obtained for 16 individuals, and no H3 sequence obtained for 15 individuals.  But we can certainly call it 240 for round numbers.

[9] The Genbank accession number of the “clade 14” Shades Creek 16S sequence is KT164167.  Blasting that sequence against the 16S sequence from a random clade 15 Shades Creek snail (KT164165) yielded an identity of 94%.  Blasting KT164167 against a random Clade 14 16S sequence from the Cahaba River at US52 (KT164235) yielded an identity of 99%.

[10] In the first paragraph of their discussion, our good friends Nathan Whelan and Ellen Strong wrote, “We show for the first time in pleurocerids that there is complete congruence in phylogenetic signal between the COI and 16S mitochondrial genes.” They have completely neglected to cite the Dillon & Frankis (2004) paper, either here or anywhere else in their work, even though Dillon & Frankis was heavily-cited by Dillon & Robinson (2009), of which they obviously were aware.  I am sure this was just a simple oversight.

[11] In fact, we did find a cryptic species in the same (2011) allozyme survey that forms the basis of the recent note by Dillon & Robinson [7].  Put another bookmark here – we’ll come back to this later, as well.

[12] The primary reason it has continued to be so difficult (in our experience) to publish reports of mitochondrial superheterogeneity is the immediate (and passionate) objection of reviewers that we have overlooked cryptic species, despite our very good allozyme evidence to the contrary.  That climate now seems to be improving.

Friday, February 5, 2016

Marstonia letsoni, Quite Literally Obscure


Editor’s Note.  This is the second essay of a two-part series on an enigmatic population of hydrobiids in Lake St. Clair, on the US/Canada border east of Detroit.  It will only make sense if you’ve read my post of [19Jan16] first.

In late September of last year I began to mentally outline a short series of essays to catch us up on invasive species news, such as have become a regular feature of this blog.  And I thought to myself, what better way to reintroduce the subject of introduced species than to tell the story of an unidentified hydrobiid snail from Lake St. Clair, which must certainly represent an invasion not heretofore recognized by anybody but yours truly, Rob Dillon?

I don’t know why I picked up Bob Hershler’s (1994) Pyrgulopsis monograph [1] and began leafing through it on Friday morning, 9/25.  I knew that Bob had reviewed 11 Eastern species as well as 54 Western species of Pyrgulopsis in that most thorough and useful work.  I also knew that Bob had teamed up with Fred Thompson in 2002 to “resurrect” Baker’s generic nomen “Marstonia,” transferring those 11 Eastern Pyrgulopsis species into it [2].  In the last 20 years I have continued to find Bob’s 1994 Pyrgulopsis monograph very helpful as I have stomped around Georgia and Tennessee, paying respects to endemic or narrowly-restricted populations of what are now called Marstonia agarhecta, Marstonia arga, Marstonia halcyon, Marstonia ogmorphaphe, and others.

I thought I knew the work pretty well.  But on the morning of 9/25, Bob’s image of a 2.8 mm “Pyrgulopsis” letsoni from a small lake west of Pontiac, Michigan jumped out and bit me like a snake.  There are two species of Marstonia inhabiting Great Lakes drainages, not just the one!  And everything about M. letsoni fit our Lake St. Clair unknowns: the shell morphology, the diminutive adult size, the range, indeed the very obscurity of it.

“Amnicola” letsoni was described by Bryant Walker [3] in 1901 from Pleistocene deposits on the banks of the Niagara River in upstate New York [4].  The first living specimens were not collected until 1943, however, when E. G. Berry dug the silt and mud out of the “honeycombed cavities” in rocks from the bed of a temporarily-dewatered stretch of the Huron River above Ann Arbor, and washed the goop through a sieve [5].  The species first known as Amnicola letsoni, and then later as Pyrgulopsis letsoni, and now as Marstonia letsoni is, quite literally, obscure [6].

Amnicola/Pyrgulopsis/Marstonia letsoni is so obscure that all the index entries under “letsoni” in Burch’s [6] North American Freshwater Snails direct the user to incorrect pages.  (Burch’s little line drawing of a Pyrgulopsis letsoni shell is hidden on the bottom of page 238.)  Arthur Clarke [7] did mention (but did not figure) Pyrgulopsis letsoni under Marstonia decepta (page 60), only venturing so far as to suggest that letsoni “may live in Canada.”

But returning to the morning of 9/25, especially compelling to me was the penial morphology.  In his message of 8/28, Bob had described the penis of the Lake St. Clair unknowns as “bifurcate.”  But I have never personally seen a Marstonia penis I would describe in that fashion.  Nymphophiline hydrobiids, such as Marstonia, bear a penis with a single duct plus a glandular lobe.  Typically the glandular lobe is larger than the duct that transmits the sperm, so that the overall nymphophiline penial morphology is bladelike, with a little “penial filament” hanging out along the margin.  E. G. Berry’s figure of the Marstonia lustrica penis at left below is typical.  The adjective “bifurcate” is not called to mind.


But Berry’s [5] figure of the penis of Marstonia letsoni illustrated a glandular lobe much smaller than any I have ever seen in a Marstonia – smaller than the duct that bears the sperm.  The overall morphology of the letsoni penis shown at right above might indeed be described as “bifurcate.”

So on 9/26 I swapped a couple fresh emails with Bob Hershler, asking for clarification on the penial morphology of the Lake St. Clair unknowns, and suggesting Marstonia letsoni as a possible identification.  And here is what Bob said: “I did think of letsoni in this context, but, again, I will need to see more specimens of the mystery snail to nail it down.”  A cautious worker is our good buddy Bob.

Well, those of you who know me, know that I think of evolutionary biology as a science – the construction of testable hypotheses about the natural world.  And the names I assign to populations of freshwater gastropods are hypotheses.  Sometimes they are weak hypotheses, and sometimes they are strong hypotheses, but Rob Dillon’s identifications are never “nailed down.”

So I composed the present two-part essay series in early October, firmly and boldly hypothesizing that the Lake St. Clair unknowns are Marstonia letsoni (Walker 1901), and prepared to go to press.  But there is yet one final twist in this story.  Our good buddy Ron Griffith asked me to delay release of the news as he was (even at that moment, in October) writing up a note for formal publication.

Ron reported that he had access to historical data from Lake St. Clair benthic samples, suggesting that the rise and spread of our M. letsoni population seems to be a fairly recent phenomenon.  In other words, M. letsoni may be a native invasive.  Might its range currently be demonstrating a significant expansion?  Ron also wanted time to send additional samples to Bob H., to try again for that "nailing down" thing.

So as most of you will have subsequently noticed, I wrote essays on three other invasive species topics for the months of October, November, and December.  And in the interim, Ron G. and Bob H. have been busy with the malacological hammer, confirming our identification even to Bob’s exacting standards.  And now at long last, nine months after that first little sample of tiny snails spilled under my dissecting scope in Milwaukee, our six-member team has reached consensus on an identification of M. letsoni.  Cradled in the metropolitan Detroit/Windsor area, bathed in one of the world’s busiest waterways, and yet remaining quite surprisingly, quite literally, obscure.


Notes

[1]  Hershler, R. (1994)  A review of the North American freshwater snail genus Pyrgulopsis (Hydrobiidae).  Smithsonian Contributions to Zoology 554: 1-115.

[2] Thompson, F. G. & R. Hershler (2002)  Two genera of North American freshwater snails: Marstonia Baker, 1926, resurrected to generic status, and Floridobia, new genus (Prosobranchia: Hydrobiidae: Nymphophilinae).  The Veliger 45: 269 - 271.

[3] For my tribute to Bryant Walker, see:
  • Bryant Walker’s Sense of Fairness [9Nov12]
[4]  Walker, B. (1901)  A new Amnicola.  Nautilus 14: 113-114.

[5] Berry, E. G. (1943)  The Amnicolidae of Michigan: Distribution, ecology, and taxonomy.  Misc. Publ. Mus. Zool. Univ. Mich. 57: 1 – 68.

[6] Burch, J. B. (1989)  North American Freshwater Snails.  Malacological Publications, Hamburg, MI.  365 pp.

[7] Clarke, A. H. (1981)  The Freshwater Molluscs of Canada.  National Museum of Natural Sciences, Ottawa.  446 pp.