Molecular phylogeny and DNA barcoding confirm cryptic species in the African freshwater oyster Etheria elliptica Lamarck, 1807 (Bivalvia: Etheriidae

Molecular phylogeny and DNA barcoding confirmcryptic species in the African freshwater oyster Etheriaelliptica Lamarck, 1807 (Bivalvia: Etheriidae)

CURT L. ELDERKIN1*, CATHARINA CLEWING2, OSCAR WEMBO NDEO3 andCHRISTIAN ALBRECHT2

1Department of Biology, The College of New Jersey, Ewing, NJ 08638, USA2Department of Animal Ecology and Systematics, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32 IFZ, 35392, Giessen, Germany3Hydrobiological Department, Faculty of Sciences, University of Kisangani, Kisangani, OrientalProvince, DR Congo

Received 2 February 2015; revised 31 October 2015; accepted for publication 31 October 2015

Recent molecular approaches to taxonomy have led to a steady increase in the identification of cryptic species.Within the Etheriidae, the species Etheria elliptica (freshwater oyster) is widespread and common and exists inmost of the major African drainages. Within the African freshwater ecosystems, there are major threats tobiodiversity and cryptic species complicate conservation strategies; unknown species exist and no conservationstatus has been assigned. Our objective here was to determine if E. elliptica from several locations in the Congodrainage are correctly classified as representing a single species. We analysed the genetic diversity at twomitochondrial loci (COI and 16S) and two nuclear loci (H3 and 28S), and estimated evolutionary relationships usingphylogenetic and DNA barcoding techniques. Bayesian inference yielded three cryptic species of Etheria, andmismatch analysis revealed discrete differences between the cryptic species. We identified three cryptic specieswithin these collections, and evidence indicates that the third species may resolve further with more sampling. Inconclusion, the taxonomic history of E. elliptica makes finding cryptic species unsurprising. However, molecularstudies such as this may finally help to resolve the number of species within this genus. © 2016 The LinneanSociety of London, Biological Journal of the Linnean Society, 2016, 00, 000–000.

ADDITIONAL KEYWORDS: 16S – 28S – biodiversity – COI – Congo drainage system – conservation –H3 – Mollusca – sub-Sahara Africa.

INTRODUCTION

Molecular approaches to taxonomy have led to asteady increase in the classification of so-called cryp-tic species (Bickford et al., 2007), a problem thatmay pre-date the Linnaean classification system(Winker, 2005). In fact, the number of cryptic speciesmay be directly proportional to the total number ofspecies in all major classes and cryptic species areespecially prominent in marine Metazoans (Knowl-ton, 2000). Prior to molecular techniques, evolution-ary relationships among bivalves were determinedusing morphological species concepts (Bickford et al.,2007). The advent of molecular techniques revealed

that some morphological characters, such as shellstructures in bivalves, are highly polymorphic andchange due to environmental and population densityeffects (Lydeard & Lindberg, 2003). The applicationof modern molecular techniques has revised taxo-nomic relationships at all levels, and illuminatedcryptic evolutionary relationships. Establishing thetaxonomic status among Metazoans is particularlyneeded because during the past century conservationissues have outpaced the taxonomic revision of spe-cies (Lydeard & Roe, 1998; Bogan & Roe, 2008). Onearea where conservation needs are greatly outpacingtaxonomic revisions is for African freshwater ecosys-tems (Dudgeon et al., 2006; Graf et al., 2011).

African freshwater ecosystems are under pressuredue to environmental threats, especially in biodiversity*Corresponding author. E-mail: [email protected]

1© 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, ��, ��–��

Biological Journal of the Linnean Society, 2016, ��, ��–��. With 4 figures.

mailto:[email protected]

hotspots such as the African Rift Lakes and Mada-gascar (Dudgeon et al., 2006). Furthermore, it wasrecently noted that the majority of African freshwa-ter molluscs are either listed as endangered or thereis little information available regarding their status(Graf et al., 2011). African freshwater bivalves com-prise a diverse group, where within the order Union-ioda there are three families present: Iridinidae,Unionidae and Etheriidae. Based on thebiogeographical distribution of African fauna, Africandrainages have been divided into freshwater ecore-gions for conservation (Thieme et al., 2005), andbivalve diversity mostly corresponds to thesedelineations (Graf & Cummings, 2011) and generallyreflects the availability of surface water (Hauffeet al., 2014).

The Etheriidae are freshwater oysters includingthe African freshwater oyster Etheria elliptica,whose range includes multiple African hotspots andecoregions. Etheria elliptica is able to cement itslower valve to any hard substrate, including otherbivalves (Pilsbry & Bequaert, 1927; Yonge, 1962;Mandahl-Barth, 1988; Bogan & Hoeh, 2000). Thiscementing behaviour has led to the loss (in adults) ofthe foot for this and other cementing bivalve species.Either valve may cement and the lower valve is usu-ally thicker (Mandahl-Barth, 1988). The structure ofthe nacre is unique with a ‘blistered or cellular’structure to the shell (Pilsbry & Bequaert, 1927) andthe shell lacks internal teeth or other structures.The periostracum is yellow to brown (mostly brownin adults) and can have long tube-like structures onthe shell; however, often the shell is badly worn andthe periostracum and any external structures areabsent. Shell shape is highly polymorphic and canrange from round to highly elongate, and the growthpattern of the shell can vary widely (Yonge, 1962;Mandahl-Barth, 1988). Etheria elliptica is dis-tributed widely throughout Africa (Fig. 1), includingmultiple drainages in southern Saharan Africa, theNile River drainage, and in highly impacted areasincluding the African Rift Lakes and northern Mada-gascar (Mandahl-Barth, 1988). All freshwaterbivalves are considered indicator species becausemost respond negatively to habitat fragmentationand pollution (Lawler et al., 2003); for example,E. elliptica populations in Madagascar have recentlygone extinct (Van Damme, 2011). Also, E. elliptica isfurther impacted because across its range this spe-cies was harvested for food and income, a practicethat continues today (Pilsbry & Bequaert, 1927;Ampofo-Yeboah, Owusu-Frimpong & Yankson, 2009).

For Etheria, its highly polymorphic shell structure,lack of internal structures, unusual growth patternsand clumping behaviour have led to taxonomic diffi-culty based on chonchological analysis alone. The

earliest taxonomic assignments of Etheria spp.(= Aetheria) were given by Lamarck (1807) in whichhe described four species incorrectly assigned to theIndian Ocean. Over the next century, four (or moredepending on the source) species were described,along with several subspecies (Pilsbry & Bequaert,1927; Yonge, 1962; Van Damme, 2011; Graf & Cum-mings, 2013). Early in the 20th century, all Etheriaspecies were lumped into the single species E. ellip-tica by von Martens (1897), later by Germain (1907)and by Anthony (1907), with E. cailliaudi andE. tubifera remaining species/sub-species designa-tions until the mid-20th century (as cited in Yonge,1962; Pilsbry & Bequaert, 1927). Etheria ellipticawas accepted as the sole African freshwater oysterfor half a century; however, it was recently sug-gested that a second Madagascan Etheria species isprobable (Graf & Cummings, 2007). Moreover, in arecent study, reproductive activity among femaleswas variable enough to conclude that multiple Ethe-ria species may exist in a single population (Bauer,2013).

With its taxonomic history, we predict that (1)there are cryptic species within the genus Etheria,(2) the application of molecular techniques is overduefor this genus and (3) sister species may exist at theregional and local level. Our objective was to take amolecular phylogenetic approach and use DNA bar-coding analysis to review the taxonomic status ofthis widespread African species. The Madagascanpopulations have been recently described as ‘likely tobe extinct’ (Van Damme, 2011). Therefore, a molecu-lar revision is even more imperative and cryptic spe-cies should be identified before extinctions ofunidentified taxa occur (Bickford et al., 2007). Wealso take a phylogeographical approach to examinethe distribution of genetic diversity across the land-scape. Optimally, we would sample as many repre-sentative areas as possible. However, African tissuecollections are not extensive and those that exist areusually preserved in solutions inappropriate formolecular DNA analysis. Therefore, we take a pre-liminary approach and use these analyses to identifyany potential cryptic species within our current,albeit limited, tissue collections.

MATERIAL AND METHODS

Etheria elliptica were collected from two sites on theChambeshi River, which forms the easternmost partof the Congo drainage basin in Zambia and threesites at the Congo River, DR Congo (see Table 1 andFig. 1). Up to 16 specimens were collected from eachsite. Individuals were collected by hand, by sight orby feel. Voucher specimens of Chambeshi River

© 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, ��, ��–��

2 C. L. ELDERKIN ET AL.

samples are deposited at the Academy of NaturalSciences, Philadelphia (Lot 419703, 419710), and theIllinois Natural History Survey mollusc collection(Lots 33665, 33671). The remaining vouchers aredeposited at the Systematics and Biodiversity collec-tion of the University of Giessen (UGSB 9365, 9366,16200, 16204). All individuals were destructively

sampled. Tissue was preserved in 80% ethanol andstored at �20 °C.

Total DNA was extracted from a small amount ofmantel tissue of individual oysters using a Qiagentissue extraction kit according to the manufacturer’sspecifications or the CTAB protocol described byWilke et al. (2006). For the phylogenetic analyses,

Figure 1. Map of the distribution range of Etheria elliptica and the five collection sites (1–5) on the African continent.


CRYPTIC DIVERSITY IN ETHERIA ELLIPTICA 3

we obtained sequences from four genes: (1) mitochon-drial cytochrome c oxidase subunit I (COI), (2)mitochondrial large ribosomal subunit (mtLSU rRNAor 16S), (3) nuclear large ribosomal subunit (nLSUrRNA or 28S) and (4) nuclear histone 3 (H3). Modi-fied primers were used for the amplification of COI:22me (50-GGTCAACAAATCATAAAGATATTGG-30)and 700dy (50-TCAGGGTGACCAAAAAATCA-30;modified from Folmer et al., 1994). We used the fol-lowing standard primers for the amplification of 16S,H3 and 28S: 16Sar-L and 16Sbr-H (Palumbi et al.,1991), H3F and H3R (Colgan, Ponder & Eggler,2000), and D23F and D6R (Park & Foighil, 2000),respectively. For COI, PCRs followed the pro-gramme; 94 °C for 2 min; five cycles of 94 °C for30 s, 30 °C for 30 s and 74 °C for 45 s; 50 cycles of94 °C for 30 s, 52 °C for 15 s and 74 °C for 1.5 min;with a final extension of 74 °C for 2 min. For 16S,H3 and 28S, PCRs followed the programme: 94 °Cfor 2 min; followed by 30 cycles of 94 °C for 30 s,55 °C for 30 s and 74 °C for 45 s. PCR products wereisolated on a 1.5% agarose gel and DNA wasextracted using a Qiagen gel extraction kit,

according to the manufacturer’s specifications. Theproducts were then amplified in a cycle sequencingreaction using the same primers, and forward andreverse sequences were visualized on a Beckman-Coulter CEQ-8000 or an ABI 3730 XL sequencer.

PHYLOGENETIC AND MOLECULAR CLOCK ANALYSES

This study includes new genetic data for a total of 25specimens (24 COI, 16 16S, 12 H3 and seven 28Ssequences; see Table 1 for details and accession num-bers). Additionally, previously published sequencesof E. elliptica were downloaded from GenBank (COI:DQ241803, AF231742). Aspartharia pfeifferiana(COI: KC429107, 16S: KC429264, H3: KC429184,28S: KC429445) and Velesunio ambiguus (KC429106,KC429263, KC429183, KC429444) were used as out-groups. Prior to the individual alignment of all frag-ments, the first base pairs behind the 30 end of eachprimer (difficult to read) were removed leaving a432-bp-long COI, 405–410-bp-long 16S, 309-bp-longH3 and 710–747-bp-long 28S fragment. The protein-coding COI and H3 sequences were aligned by eye.

Table 1. List of studied specimens including detailed locality information, specimen ID number, taxon (Etheria sp. A,

sp. B, or sp. C) and GenBank accession numbers

River Site name Latitude (°N) Longitude (°E) ID no. Taxon

GenBank accession no.

COI 16S H3 28S

Chambeshi Mbasuma

Ranch

�10.010 32.124 E051 sp. A KT964358 KT964334 KT964348

E052 sp. A KT964358 KT964335

E053 sp. B KT964360

E054 sp. A KT964358 KT964336 KT964350

E056 sp. B KT964360 KT964353 KT964337 KT964342

E057 sp. B KT964361 KT964354 KT964338 KT964349

E058 sp. B KT964363 KT964339

E059 sp. A KT964358 KT964351

E060 sp. A KT964358 KT964352

E062 sp. A KT964358 KT964351

E063 sp. A KT964358 KT964351

Chambeshi Sawfa

Pontoon

�10.852 31.167 E096 sp. C KT964367 KT964355

E097 sp. B KT964360

E102 sp. C KT964355 KT964340 KT964345

R200 sp. B KT964360 KT964353

R201 sp. B KT964360 KT964353

R202 sp. B KT964360 KT964353

R203 sp. C KT964364 KT964356 KT964341

R205 sp. B KT964360 KT964353

R206 sp. B KT964360 KT964353

R207 sp. B KT964362 KT964353

Congo Engengele �2.993 22.663 CO664 sp. C KT964366 KT964341 KT964346

CO719 sp. C KT964357 KT964356 KT964341

Congo Kindu 1 �2.964 25.929 CO407 sp. A KT964365 KT964343 KT964347

Congo Kindu 2 �2.956 25.930 CO415 sp. A KT964359 KT964342



http://www.ncbi.nlm.nih.gov/nuccore/DQ241803

http://www.ncbi.nlm.nih.gov/nuccore/AF231742

http://www.ncbi.nlm.nih.gov/nuccore/KC429107








http://www.ncbi.nlm.nih.gov/nuccore/KT964358



























































For alignment of the non-coding 16S and 28S frag-ments, we used the multiple sequence alignmentprogram MAFFT (default settings, http://www.ebi.a-c.uk/Tools/msa/mafft; Katoh & Toh, 2008) resultingin a final alignment with a length of 417 and 747 bp,respectively. All partitions were tested for nucleotidesubstitution saturation using the test of Xia et al.(2003) implemented in DAMBE version 5.2.73 (Xia &Lemey, 2009). The test showed limited saturation forall partitions even under the assumption of an asym-metrical tree. The final concatenated dataset con-sisted of 24 individuals/specimens (including the twooutgroup taxa). Haplotype numbers (unique) were:COI = 28 (15), 16S = 23 (15), 28S = 9 (7) andH3 = 14 (8), including the outgroup taxa; the finalconcatenated dataset consisted of 24 specimens.Missing fragments were filled with N.

The phylogenetic tree was reconstructed usingBayesian inference implemented in the softwarepackage MRBAYES version 3.1.2 (Ronquist &Huelsenbeck, 2003) with the following parameters:nchain = 4, ngen = 4 000 000, samplefreq = 100,temp = 0.1. Prior to this analysis, JMODELTESTversion 0.1.1 (Posada, 2008) was used to find the bestfit model of sequence evolution for the individualdataset based on the corrected Akaike informationcriterion. For the 28S and H3 partitions the programselected the GTR+I model. For the COI and 16S par-titions, the HKY+I model and the GTR+G modelwere selected, respectively. Monitored in TRACERversion 1.5.0 (Drummond & Rambaut, 2007), thecombined set of trees showed both high ESS (effec-tive sample size) values (> 3000 for all parameters)and a smooth frequency plot. For the consensus tree,the first 25% of the sampled trees (N = 10 000) werediscarded as burn-in.

Additionally, the COI partition was used for molec-ular clock analyses. Depending on the assumption ofhomogeneous or heterogeneous substitution ratesamong the branches, either a strict clock or a relaxedclock analysis can be performed. To test whether thestrict clock model can be accepted, a Bayes factoranalysis was conducted. Strict and relaxed clockanalyses were performed using BEAST version 1.7.5(Drummond & Rambaut, 2007), with the followingparameters: site model = HKY+I, clock model = (1)strict clock/(2) relaxed lognormal clock (for both, arate of min. = 0.0124 and max. = 0.0157 was appliedreferring to the models HKY and HKY+I+G, respec-tively, as a substitution rate for the model HKY+I isnot provided by Wilke, Schultheiß & Albrecht, 2009),speciation = Yule process, ngen = 20 000 000, andlog = 1000. The Bayes factors (log10 Bayes factor)were calculated in TRACER (parameters: likelihoodtrace = treeLikelihood, bootstrap replicates = 1000).We used the thresholds proposed by Kass & Raftery

(1995): 0–3 (positive support), 3–6 (strong support)and > 6 (decisive support).

BARCODING ANALYSIS, MOLECULAR DIVERSITY AND

POPULATION STRUCTURE

The Consortium for the Barcode of Life (Fr�ezal &Leblois, 2008) has developed a standardized molecu-lar identification system (Hebert, Cywinska & Ball,2003) to rapidly sort among species that are morpho-logically indistinguishable. This DNA barcode is ashort (about 650 bp) fragment of the COI. Using a432-bp fragment of the Etheria COI gene, sequenceswere analysed in ARLEQUIN version 3.5 (Excoffier& Lischer, 2010) to examine base pair differences.

Mismatch distributions, which display pairwisenucleotide differences, were used to visualize thegenetic differences within and between Etheria sam-ples and determine whether fixed nucleotide differ-ences were present. Previous studies have used thisanalysis to determine cryptic species, where a mis-match analysis will yield a bi-modal distributionwith a ‘Barcoding Gap’ between species (Meyer &Paulay, 2005; Alexander et al., 2009; Elderkin et al.,2012; Pri�e & Puillandre, 2014). We also used ARLE-QUIN to calculate preliminary estimates of bothgenetic diversity and pairwise estimates of FST

within each clade. However, some of these estimateswere not possible due to the limited numbers of sam-ples that successfully yielded COI and 16Ssequences. We did not attempt population or diver-sity estimates using H3 and 28S due to the low num-ber of samples sequenced. We used TCS version 1.21(Clement, Posada & Crandall, 2000) to obtain a par-simony network of all COI sequences for each of thethree species (with a 95% root probability). Ambigu-ity in the network (loops or circular pathways) wasresolved using a coalescence model as outlined byFetzner & Crandall (2003).

RESULTS

The phylogenetic analysis revealed three highly sup-ported clades that putatively correspond to three dis-tinct species of Etheria (see Fig. 2A, labelled Etheriasp. A, Etheria sp. B and Etheria sp. C) with a Baye-sian posterior probability (BPP) of 0.96 and 1.00(Fig. 2A). Two smaller clades had significant branchsupport within Etheria sp. C. However, these repre-sented only a few individuals each and did not yieldconsistent results using barcode analysis (see below).The two sequences from GenBank nested within twoseparate clades (Etheria sp. B and sp. C). Within theparsimony network each clade was separated by asignificant number of fixed site differences (see



http://www.ebi.ac.uk/Tools/msa/mafft

http://www.ebi.ac.uk/Tools/msa/mafft

Fig. 3, Table 2). The parsimony networks revealedthat most species were collected at more than onesite in the Congo River drainage (Fig. 3). Etheria sp.A was found at the Mbasuma Ranch location in theChambeshi River and at the two Kindu sites in theCongo River; Etheria sp. B and sp. A were found atboth Chambeshi River collection sites and the onlyspecies found at the Engengele Congo location wasEtheria sp. C (Fig. 3).

Bayes factor analyses resulted in positive support(0.63) for the relaxed lognormal clock model and thusslightly favoured a heterogeneous substitution rateamong branches. The clock analyses revealed diver-gence age estimates ranging from 0.45 Myr (mean

age; 95% highest posterior density, HPD: 1.00–0.05 Myr) for the youngest clade (Etheria sp. A) to7.87 Myr (11.77–4.43 Myr) for the first split of Ethe-ria spp. (see Fig. 2B). The age of the most recentcommon ancestor of Etheria sp. C and Etheria sp. Bis 4.99 Myr (7.45–2.75 Myr). Thereby, the age of thefirst diversification event within the clade Etheriasp. C is comparatively older than for the clade Ethe-ria sp. B (1.94 Myr; 3.29–0.91 vs. 1.15 Myr; 2.16–0.41).

DNA barcoding (COI results only) resulted in threedistinct modes in the analysis (Fig. 4) that directlycorrespond to the phylogenetic analysis (Etheria sp.A, sp. B and sp. C). The first major division is a

A

B

Figure 2. (A) Bayesian phylogram of Etheria spp. based on COI, 16S, H3 and 28S sequences. Bayesian posterior proba-

bilities are given next to the respective node. Major clades are marked with white or grey bars. Individuals with identi-

cal haplotypes are numbered in small grey boxes to the right of respective branches. The location of each specimen is

labelled as follows: 1Congo River (Engengele), 2Chambeshi River (Mbesuma Ranch), 3Chambeshi River (Sawfa Pontoon),4Congo River (Kindu 1), 5Congo River (Kindu 2) and NAnot available. (B) Simplified Bayesian phylogenetic tree of Ethe-

ria spp. under the relaxed lognormal clock model inferred only from COI. The ages of most recent common ancestors dis-

cussed in the text are illustrated including their 95% HPD intervals (blue bars). Outgroup taxa are not shown. For

detailed specimen information see Table 1.



54-bp (13%) divergence (between Etheria sp. A andsp. B), which represents single base pair mutationsat 54 fixed sites in the COI gene that can beassigned to each clade. The second major division isa 57-bp (13%) divergence (between Etheria sp. A andsp. C) and 39-bp (9%) divergence (between Etheriasp. B and sp. C). Although Etheria sp. C also yieldeda fourth mode in the analysis, the locations of thebase pair mutations were not from fixed sites andthey could not be assigned as a fourth species usingbarcode analysis.

The estimates of diversity and population struc-ture were limited by the sample size of individualsfrom each of the three clades (FST 0.08–0.73; nucleo-tide diversity 0.002–0.032). The mean number ofpairwise differences was low within species (0.2–3.0%), and high among species (2–13%). Overall, pre-liminary estimates of genetic diversity and popula-tion structure varied among clades and depended onthe molecular marker used. The highest amount ofmolecular diversity was found in Etheria sp. C inCOI (0.032) and Etheria sp. A in 16S (0.013). Simi-larly, the highest degree of population differentiation(FST = 0.79) was found in Etheria sp. C in COI andwas also high in Etheria sp. A in 16S (FST = 0.73).

Interestingly, Etheria sp. A had a low nucleotidediversity in the COI locus but was relatively diversein the 16S locus.

DISCUSSION

Our results indicate the presence of three distinct,highly divergent clades for Etheria from a single Afri-can drainage (River Congo drainage). The divergenceindicates long-term isolation between these threeclades. Also, there is evidence of significant evolution-ary patterns among branches within Etheria sp. C(see below). However, further molecular analysis,including an increased number of individuals andareas sampled, is necessary to confirm these relation-ships. Given the taxonomic history of this genus, it isnot unexpected to find cryptic species (Pilsbry &Bequaert, 1927; Mandahl-Barth, 1988), and similarcryptic species have been found in the freshwatermussel Anodonta anatina (Froufe et al., 2014). Cryp-tic species occur most often when chemosensory sys-tems are more important than the visual senses(Mayr, 1963). Etheria are similar to marine molluscsin that they may depend largely on chemical signals

Figure 3. Map of the collection locations of Etheria elliptica from two sites on the Chambeshi River, Zambia, and three

sites on the Congo River, DR Congo (left). Parsimony networks using COI sequences of the three putative species of

Etheria (right); size of the circle represents the number of individuals with identical haplotypes (numbers > 2 shown

below the ID number). To the left pie charts represent the distribution of species among the three collection sites; col-

ours correspond to the colours of the haplotype networks to the right.



Table

2.Listof

fixed

sitesforthenuclea

rhistone3(H

3)andthenuclea

rlargeribosom

alsu

bunit

(28S)

Primer/

IDLocation

H3

69

24

63

72

99

141

150

159

168

183

186

201

240

282

E051

CT

TT

AA

TT

CC

CA

CG

T

E052

..

..

..

..

..

..

..

.

E054

..

..

..

..

..

..

..

.

21415

..

..

..

..

..

..

..

N

21407

..

..

..

..

..

..

..

N

E056

TC

AC

GG

GC

.T

.C

TA

.

E057

TC

..

GG

GC

.T

.C

.A

.

E058

TC

..

GG

GC

.T

AC

TA

.

R203

TC

A.

GG

G.

GT

..

TA

.

E102

TC

.G

GG

..

T.

..

AC

7664

NC

NN

GN

N.

NT

N.

NN

N

7719

NC

NN

GN

N.

NT

N.

.N

N

28S

68

69

70

72

90

98

128

133

140

142

163

182

184

200

218

224

227

231

234

249

297

348

357

375

395

407

411

427

429

592

600

711

E051

TG

TT

GA

TG

CC

CC

GG

GG

TA

TG

TC

CA

GC

AC

CC

CC

1407

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

E054

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

..

E056

..

GN

AA

NA

..

T.

TA

AN

GT

CA

N.

TN

TT

.A

TT

.T

E057

..

NN

AA

NA

.N

T.

TN

AA

NT

NN

C.

TN

TT

.A

.T

.T

E102

GC

GC

AC

AA

TT

GT

.A

.G

TC

AC

T.

CT

TG

.T

TG

T

7664

GC

GC

..

C.

..

..

..

A.

GT

C.

C.

.C

..

..

..

.T

Colou

rscorrespon

dto

thewhiteandgreybox

eslabellingdifferentputativesp

eciesof

Etheria

inFig.2.



for reproduction (Palumbi, 1994). Also, because theseare lentic and lotic species, behaviours that mayeasily differentiate species remain largely unseen andunstudied by scientists (Knowlton, 2000). Until now,Etheria has not been the subject of a molecular studyregarding cryptic species. Although these results arepreliminary, the degree of differentiation among indi-viduals indicates at least three distinct Etheria spe-cies can be found with limited sampling and within asingle African drainage.

The mismatch distribution supports our hypothesisthat DNA barcoding can be used to differentiatethese three cryptic Etheria species. We found fixeddifferences in the mitochondrial COI gene, and weresuccessful in identifying three species of adult speci-mens collected from the Congo River drainage thatcould not be distinguished morphologically. DNAbarcoding analysis is dependent on the assumptionof a significant gap between intraspecific andinterspecific DNA variation (Meyer & Paulay, 2005),

especially for identifying cryptic species. Previousresults of barcoding analysis have been inconclusivewhen investigators encountered considerable overlapbetween intra- and interspecific variation (Meyer &Paulay, 2005; Alexander et al., 2009). However, ourresults overwhelmingly point to three species withno overlap. Our results confirm the existence of asignificant gap between Etheria COI sequences andsupport that DNA barcoding can be used to distin-guish these cryptic species.

Etheria elliptica and all freshwater bivalves dis-perse using a unique juvenile stage that depends ona fish host. In the Unionidae, this life history cycle iswell understood; glochidia larvae briefly attacheither to the gills or fins of fish hosts (Hoggarth,1999). Similar to the Unionids, Etheria spawningappears to be synchronous and the female brieflybroods offspring in her gills (Bauer, 2013). However,Etheria offspring are released as lasidium larvae andfrom there little of their life history and dispersal

Figure 4. Mismatch distribution displaying the number of pairwise nucleotide differences among all individuals at the

COI locus. Note the tri-modal nature of the distribution with a considerable barcoding gap between different clades of

Etheria elliptica. The two extreme modes to the right represent a 9% (Etheria sp. B vs. sp. C) and a 13% (Etheria sp. A

vs. sp. B and sp. C) difference in individuals, respectively. However, the smaller mode represents differences within

Etheria sp. C (see Results and Discussion).



ability is known (Bogan & Roe, 2008; Bauer, 2013).If Etheria are similar to other lasidium-producingbivalves such as Anodontites trapesialis (Mycetopodi-dae) they briefly encyst in the outer skin of a fishhost and are thereby dispersed (Silva-Souza & Eiras,2002). However, the length of the infection and thehost fish dispersal ability are unknown (Bogan &Roe, 2008; Bauer, 2013). In our estimates of pairwisepopulation structure, Etheria populations were rela-tively isolated compared with other freshwaterbivalves: Amblema plicata, FST = 0.18; Actinoniasligamentina, FST = 0.16; Elliptio dilatata, FST = 0.62(Elderkin et al., 2006, 2008); Velesunio spp.,FST = 0.17–0.68 (Hughes et al., 2004). The amount ofmolecular diversity within each species (p) was rela-tively low compared with other freshwater bivalvespecies: Actinonias ligamentina, p = 1.71–6.11; Ellip-tio dilatata, p = 0.15–6.50 (Elderkin et al., 2008). InElliptio dilatata and Velesunio spp., the relativelyhigh FST values indicated low gene flow among popu-lations, and both studies indicate restricted host fishdispersal as a cause. The Congo River is one of thelargest rivers on Earth, and has many geographical(and hydrological) barriers to fish dispersal. Thesecharacteristics may have facilitated speciation insome Congo River fish species (Markert, Schelly &Stiassny, 2010).

The amount of sequence divergence among thethree species was very high; however, we are unableto assign a preliminary taxonomic designation. Theamount of genetic divergence at COI was on the wholegreater than those found among other species of fresh-water bivalves in Unionidae: Coelatura gabonenesisvs. C. aegyptiaca = 9% (Whelan, Geneva & Graf,2011), Elliptio dilatata vs. E. complanata = 7%(Elderkin et al., 2008; C. L. Elderkin, unpubl. data)and among Unio delphinus vs. U. pictorum 4.44%(Khalloufi et al., 2011). Our estimates were more simi-lar to comparisons among genera of other freshwaterbivalves in Etheriidae: Acostaea rivoli vs. Etheriaelliptica (14%); Iridinidae: Chambardia wahlbergi vs.Aspatharia pfeifferiana (13%); Unionidae: Lampsilissp. vs. Obliquaria reflexa 12% (Roe, Hartfield &Lydeard, 2001); and among species in the Hyriidae:Velesunio spp. 9–12% (Baker et al., 2003). However,estimates using 16S data were similar to comparisonsbetween other freshwater congeneric species: Vele-sunio spp. = 1–12% (Baker et al., 2004), and Lamp-silis spp. 4–10% (Roe et al., 2001).

The molecular clock estimates among the Etheriaspecies correspond to significant geological and cli-matic changes on the African continent. Approxi-mately 12–5 Mya (corresponding to the differencesbetween Etheria sp. A and sp. B/C) a land bridgeformed between North and South America perma-nently changing global ocean currents. Following this

event, the African continent began to cool with adrier climate, and vegetation changing from wood-land to savanna. Also around 6–4 Mya (correspondingto the differences between Etheria sp. B and sp. C),west African uplift increased, rapidly changing thewind and weather patterns over eastern African(Gani, Gani & Abdelsalam, 2007). The African Riftvalley is within the easternmost range of Etheria.During this period, mammals including hominidsappear to have evolved rapidly due to climate change(Lorenzen, Arctander & Siegismund, 2006; Reynolds,2007). In the same period, many basins suitable forlakes formed in the African Rift valley (Maslin &Christensen, 2007) providing areas for range expan-sion of freshwater organisms. The dry climate of theRift Valley was also punctuated by some (short) wetperiods. This fluctuation may have facilitated long-term isolation followed by rapid dispersal in freshwa-ter organisms. These climate patterns may havehelped to facilitate speciation among populations ofEtheria. Certainly the timeline for most recentbranches within Etheria sp. B and sp. C correspondto more familiar examples of African freshwater spe-ciation. These branches correspond to a time whenAfrican lakes experienced low water 1.6–1.0 Mya.Recent studies found that African cichlids and fresh-water gastropods rapidly diversified into multiplespecies during this same period (Sturmbauer et al.,2001; Seehausen, 2006; Schultheiß et al., 2011, 2014).

Etheria sp. A and sp. C are found both in the mainchannel of the Congo River and the eastern tributaryof the Chambeshi River. However, in the same drai-nage this distribution represents several thousandkilometres of separation between populations. CongoRiver Etheria sp. A samples were only a single basepair apart from Chambeshi samples. However,within Etheria sp. C we found significant branchsupport separating the Congo samples from theChambeshi River samples. Also, Etheria sp. C had aunique within-group mode in the barcoding analysis.Considering the above evidence, we suggest furthersampling and additional molecular markers for indi-viduals in the clade labelled Etheria sp. C, as thisbranch may resolve further and yield additional spe-cies of Etheria.

CONSERVATION IMPLICATIONS

Freshwater habitats and the species that occupythose habitats are of primary conservation concerndue to specific environmental issues in these areas(Dudgeon et al., 2006). Freshwater habitats areunder increasing pressure from habitat degradation,and the evolution and ecology of organisms thatoccupy these habitats are poorly understood com-pared with terrestrial species (Strayer, 2006). Afri-



can freshwater habitats are no exception — habitatdegradation due to human population growth hasbeen and continues to be a significant problem.Therefore, to preserve endemic biodiversity, conser-vation of African freshwater molluscan fauna mustbe a priority. Currently 43% African freshwater mol-lusc species are either endangered or data-deficient(Graf et al., 2011). Cryptic species further complicateconservation strategies where species exist that havenot been described and no conservation status hasbeen assigned (Bickford et al., 2007). This study illu-minates the importance of molecular approaches thatcan further enhance our knowledge of African mol-luscan species. Etheria elliptica and African drai-nages are impacted by human population growthand it is imperative to describe new species and doc-ument their range within the continent. Further col-lections along the whole range of E. elliptica shouldbe a priority to determine the amount of geneticdiversity present within the genus. The highest pri-ority should be collections and DNA sampling ofEtheria populations in the Rift Lakes and Madagas-car as these freshwater habitats have been known toharbour endemic species and are highly impacted byhuman population growth. These results along withthe lack of information about Etheria reproduction(Bogan & Roe, 2008) make a strong case for a sys-tematic review of this genus using multiple markersalong with life history and ecological information.

ACKNOWLEDGEMENTS

We are particularly grateful to Bert Van Bocxlaer forproviding samples to this study. We thank Alex Chilala,Daniel Graf, Kevin Cummings, Anthony Geneva, Jer-emy Tieman and Jaci Kahn for field assistance. Wethank Liron Bendor and Silvia Nachtigall for assistancein the laboratory. We also thank Roland Schultheiß forhelpful assistance and stimulating conversation regard-ing the phylogenetic analysis. We are grateful for thehelpful comments of two anonymous reviewers. Tissuecollection and initial DNA analysis were supported byan ROA supplement to NSF grant DEB-054575 (DanielGraf, PI). Further support came from a Senior ResearchAward from the Fulbright Scholars Program to C.L.E.and the DFG (Deutsche Forschungsgemeinschaft) toC.A. This project was also supported by The College ofNew Jersey (USA) and Justus Liebig University Gies-sen (Germany).

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Molecular phylogeny and DNA barcoding confirm cryptic species in the African freshwater oyster Etheria elliptica Lamarck, 1807 (Bivalvia: Etheriidae