Research Article |
Corresponding author: Martin Haase ( martin.haase@uni-greifswald.de ) Academic editor: Frank Köhler
© 2016 Marlen Becker, Susan Zielske, Martin Haase.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Becker M, Zielske S, Haase M (2016) Conflict of mitochondrial phylogeny and morphology-based classification in a pair of freshwater gastropods (Caenogastropda, Truncatelloidea, Tateidae) from New Caledonia. ZooKeys 603: 17-32. https://doi.org/10.3897/zookeys.603.9144
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Morphological classification and mitochondrial phylogeny of a pair of morphologically defined species of New Caledonian freshwater gastropods, Hemistomia cockerelli and H. fabrorum, were incongruent. We asked whether these two nominal species can be unambiguously distinguished based on shell morphology or whether the taxonomic discrepancy inferred from these character types was reflected in the variation of shell morphology. Our investigations were based on phylogenetic analyses of a fragment of the mitochondrial cytochrome c oxidase subunit I, geometric morphometric analyses as well as micro computer tomography. The species presorted to morphospecies by eye overlapped in shell shape. However, statistically, all shells were correctly assigned, but not all of them significantly. Qualitatively, both nominal species can be unambiguously distinguished by the presence/absence of a prominent denticle within the shell. In the phylogenetic analyses, individuals from three populations clustered with the “wrong” morphospecies. In the absence of data from multiple loci, it was assumed for the single specimen from one of these populations that its misplacement was due to a recent hybridization event, based on its very shallow position in the tree. For the other two cases of misplacement neither introgression nor incomplete lineage sorting could be ruled out. Further investigations have to show whether the morphological overlap has a genetic basis or is due to phenotypic plasticity. In conclusion, despite their partly unresolved relationships H. cockerelli and H. fabrorum may be considered sister species, which are reliably diagnosable by the presence or absence of the denticle, but have not yet fully differentiated in all character complexes investigated.
Geometric morphometrics, hybridization, incomplete lineage sorting, introgression, morphology, shape, South Pacific, taxonomy
Conflict in phylogenetic signal between different characters, e.g. between different genes, between mitochondrial (mt) and nuclear (nc) DNA, or between genes and morphology, is commonly observed across a wide range of taxa (e.g.,
These investigations were initially the goal of the present account. Unfortunately, we could not consistently amplify ITS2 across the entire, enlarged data set. Therefore, we had to restrict this analysis to a comparison of COI-phylogeny and shell morphology. Typical H. cockerelli have a slender-conical shell whereas H. fabrorum is much broader. In addition, H. cockerelli is characterized by a prominent palatal denticle c. 1/3 whorl behind the outer lip (Figs
Most specimens examined in this study were collected in 2012 at 22 localities in New Caledonia (Table
Material investigated. Ngen, number of genetically investigated specimens; Nmor, number of morphologically investigated specimens; Pop#, population number; Pt, paratypes.
Pop# | Species | Latitude Longitude | Nmor | Ngen |
---|---|---|---|---|
1A | H. fabrorum | 22°08'59.0"S, 166°29'10.6"E | 25 | 10 |
6B | H. fabrorum | 21°48'08.0"S, 166°04'14.6"E | 36 | 9 |
8 | H. fabrorum | 21°44'32.1"S, 166°05'20,6"E | 4 | 0 |
9A | H. cockerelli | 21°44'30.9"S, 166°05'57.9"E | 3 | 2 |
10 | H. fabrorum | 21°42'55.4"S, 166°07'21.1"E | 15 | 8 |
11 | H. cockerelli | 21°48'16.8"S, 166°00'00.8"E | 26 | 7 |
13 | H. cockerelli | 21°47'30.8"S, 165°54'31.6"E | 20 | 7 |
14 | H. cockerelli | 21°47'30.8"S, 165°54'38.7"E | 11 | 8 |
15A | H. cockerelli | 21°47'24.4"S, 165°54'51.2"E | 6 | 1 |
16 | H. cockerelli | 21°47'24.4"S, 165°54'51.2"E | 10 | 2 |
17 | H. cockerelli | 21°39'52.8"S, 165°43'10.3"E | 11 | 2 |
18 | H. cockerelli | 21°39'40.6"S, 165°43'06.9"E | 22 | 1 |
25B | H. fabrorum | 21°34'15.7"S, 165°49'41.2"E | 11 | 9 |
28 | H. fabrorum | 21°31'07.4"S, 165°48'20.0"E | 11 | 10 |
30C | H. cockerelli | 21°34'21.6"S, 165°41'02.5"E | 9 | 1 |
31 | H. cockerelli | 21°33'33.5"S, 165°42'11.3"E | 7 | 2 |
32 | H. cockerelli | 21°34'55.9"S, 165°40'16.7"E | 6 | 0 |
36 | H. cockerelli | 21°38'22.1"S, 165°51'37.5"E | 9 | 3 |
38B | H. fabrorum | 21°38'09.3"S, 165°51'52.7"E | 11 | 9 |
38C | H. cockerelli | 21°38'09.3"S, 165°51'52.7"E | 10 | 6 |
39 | H. fabrorum | 21°37'56.1"S, 165°51'54.4"E | 6 | 2 |
41 | H. cockerelli | 21°38'12.3"S, 165°51'34.1"E | 5 | 0 |
46 | H. fabrorum | 21°14'30.2"S, 165°16'30.8"E | 13 | 9 |
Pt | H. cockerelli | 21°49.2’S, 166°56.6’E | 37 | 0 |
All 324 individuals were investigated for the presence/absence of a denticle 1/3 whorl behind the outer lip, which has been described as diagnostic for H. cockerelli (
For geometric morphometric investigations (
In order to visualize the morphological variation, a principal component analysis (PCA) was performed using MORPHOJ v. 1.06a (
DNA was isolated using Qiagen’s DNeasy Blood & Tissue Kit (QIAGEN GmbH; Hilden, Germany) in compliance with the manufacturer’s protocol except that we eluted only in 20 μl of AE-buffer. A fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) was amplified using the primers LCO1490 and HCO2198 (
COI sequences were edited using the software DNA BASER v. 4.16 (DNA Baser Sequence Assembler v. 4.16 2014). After addition of two outgroup sequences of H. nyo and H. andreae (see
The first two axes of the PCA comparing the morphospecies explained 76.2% of the total morphological variation. The nominal species were fairly well separated along axis 1, however, the 95% confidence ellipses were overlapping. Nineteen specimens within the area of overlap and its neighborhood as defined in Figure
A jackknife test of assignment, a cross-validation procedure, was performed a posteriori to assess the robustness of the CV axes and comprised these 305 specimens. In 10,000 replicates, 10% of the specimens (31) were randomly selected as unknown data and assigned to one of the resulting groups of the following CVA analysis based on the remaining 274 shells. 99% of the assignments were correct and significant, 1% correct and not significant, and no shell was incorrectly assigned.
In a subsequent Goodall’s F test both morphospecies were highly significantly (p < 0.001) distinguished. This test included all specimens unambiguously (p > 0.05) assigned to one of the nominal species in the previous CVA-based tests.
The μCT-based three dimensional shell surface models revealed, as expected, presence and absence of the palatal denticle in the paratype of H. cockerelli and the topotype of H. fabrorum, respectively (Figs
The phylogenetic tree reconstructions inferred very similar relationships. Figure
Phylogenetic reconstructions. A Bio-neighbor-joining tree based on COI fragment with bootstrap support values from both maximum likelihood and neighbor-joining analyses (ML/BNJ) B Maximum likelihood topology. Asterisks indicate “misplaced” individuals. In B only the “misplaced” individuals and one individual (6B.10), whose position differs considerably in both trees, are indicated. Otherwise, only the clade composition is given. Crossed branches are shortened by 50%. The outgroup was pruned from the tree. Scale bars: substitutions per site.
Conflict in phylogenetic/taxonomic signal between different characters is not uncommon among truncatelloidean gastropods, in particular in cases of recent events of speciation and young evolutionary radiations (e.g.,
As the number of individuals misplaced in the phylogenetic analyses was very low and in particular because ncDNA data were lacking, explanations for the incongruences have to remain largely speculative. However, at least for dubious shallow relationships the assumption of a recent event, which can only be introgression through hybridization, is very likely. The specimen identified as H. cockerelli from population 18 was nested among individuals allocated to H. fabrorum from three populations. Since all surrounding, more basal nodes belonged to H. fabrorum, this misplaced specimen most likely has inherited its mitochondria through introgression by hybridization of a female H. fabrorum with a male H. cockerelli in the not too distant past.
In contrast, the misplaced clades consisting of the two specimens from population 9A and the nine individuals of H. fabrorum from population 46, respectively, were connected to deeper, partly unsupported nodes. It is exactly this situation which makes the distinction of incomplete lineage sorting and hybridization difficult (
In conclusion, despite their partly unresolved relationship H. cockerelli and H. fabrorum may be considered sister species, which are reliably diagnosable by the presence or absence of the palatal denticle, but have not yet fully differentiated in all character complexes investigated. The range of H. fabrorum covers wide parts of southern New Caledonia and largely overlaps with that of H. cockerelli, which is found almost across the entire island (
In order to unambiguously identify the causes of the genetic inconsistencies an even denser sampling design as well as using more genetic markers would be required. The potential role of phenotypic plasticity can only be assessed in common garden experiments. The ambiguous genetic signal also calls for caution for barcoding (
We are indebted to Christine Pöllabauer and numerous local guides for their logistic support during field work in New Caledonia. We are grateful to Gabriele Uhl and Peter Michalik for assistance with μCT and Jakob Krieger as well as Andy Sombke with Amira. We thank Christel Meibauer for her help in the lab. Frank Köhler, Tom Wilke, and three anonymous reviewers helped to improve earlier versions of the manuscript. Financial support was received from the Deutsche Forschungsgemeinschaft (HA4752/2-1).