DNA and Morphology Unite Two Species and 10 Million Year Old Fossils

<div><p>Species definition and delimitation is a non-trivial problem in evolutionary biology that is particularly problematic for fossil organisms. This is especially true when considering the continuity of past and present species, because species defined in the fossil record are not necessarily equivalent to species defined in the living fauna. Correctly assigned fossil species are critical for sensitive downstream analysis (e.g., diversification studies and molecular-clock calibration). The marine snail genus <em>Alcithoe</em> exemplifies many of the problems with species identification. The paucity of objective diagnostic characters, prevalence of morphological convergence between species and considerable variability within species that are observed in <em>Alcithoe</em> are typical of a broad range of fossilised organisms. Using a synthesis of molecular and morphometric approaches we show that two taxa currently recognised as distinct are morphological variants of a single species. Furthermore, we validate the fossil record for one of these morphotypes by finding a concordance between the palaeontological record and divergence time of the lineage inferred using molecular-clock analysis. This work demonstrates the utility of living species represented in the fossil record as candidates for molecular-clock calibration, as the veracity of fossil species assignment can be more rigorously tested.</p> </div

The New Zealand <i>Alcithoe</i> of the last 11 million years.

<p>Fossil species are shown on the left, extant species on the right. Bars show occurrence in the fossil record in the time bins indicated, with resolution at the level of recognised New Zealand geological stages <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052083#pone.0052083-Cooper1" target="_blank">[56]</a>. Numbers by the fossil species indicate the number of discrete sampling events, as recorded in the FRED database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052083#pone.0052083-Crampton4" target="_blank">[57]</a>. <i>Alcithoe knoxi</i> is known from two collections in Tongaporutuan and is found in the extant fauna, but no fossils have yet been identified from the intervening time, as indicated by a dashed line. Abbreviations are given for New Zealand fossil record stages (Tt – Tongaporutuan, Tk – Kapitean, Wo – Opoitian, Wp – Waipipian, Wm – Mangapanian, Wn – Nukumaruan, Wc – Castlecliffian, Wq – Haweran).</p

Potential phylogenetic positions of <i>Alcithoe knoxi</i>.

<p>Morphological ambiguity results in a range of phylogenetic hypotheses for the placement of <i>A. knoxi</i>, ranging from (A) <i>A</i>. <i>knoxi</i> representing a distinct lineage (genus <i>Teremelon</i>) sister to the <i>Alcithoe</i> lineage, to (B) <i>A. knoxi</i> is a form of <i>A. wilsonae</i> nested within the modern diversity of <i>Alcithoe</i>. The node that would be calibrated at approximately 10my based on the <i>A. knoxi</i> fossils is shown (arrows). Calibration (A) would set a maximum limit of the origin of the modern <i>Alcithoe</i> at no earlier then 10mya, whereas (B) would allow the diversification of the extant <i>Alcithoe</i> to have occurred earlier.</p

Functional groups of assembled transcripts from stick insect cDNA inferred from similarity to an arthropod dataset (Reference) using evolutionary genealogy of groups: Non-supervised Orthologous Groups classification system (eggNOG).

<p>COG = Cluster of Orthologous Groups.</p

Summary of the cDNA sequences obtained from two New Zealand stick insects using the Illumina pipeline with the length distribution of transcript assemblies following trimming (at a quality of 0.001) and subsequent clustering.

<p>Summary of the cDNA sequences obtained from two New Zealand stick insects using the Illumina pipeline with the length distribution of transcript assemblies following trimming (at a quality of 0.001) and subsequent clustering.</p

Next Generation sequencing results from mRNA of two New Zealand stick insects (A).

<p>Length distributions of transcript assemblies produced from the cDNA sequence of two stick insects were similar. A log length frequency distribution plot used values rounded to 1 decimal place for the longest consensus sequence generated from each cluster. (B) Sequence divergence (measured by SNP density per nucleotide) observed when reads were mapped to ~2,600 loci (transcript assemblies). Loci without variation (SNP-free) were removed. The putative parental <i>Clitarchus hookeri</i> genome contains many loci with low allelic diversity. SNPs detected in less than 10% of the short reads were ignored but reads were included whether or not they passed the strand bias filter within VarScan. Only the longest assembled transcripts generated per cluster were included. (C) SNPs detectable on all transcript assemblies by BWA mapping to ~2,600 <i>Acanthoxyla</i> and <i>Clitarchus</i> transcript assemblies using VarScan with minimum variant frequency of 10% irrespective of strand filter results. The first violin of each color comprises all data, and the second excludes transcript assemblies with no sequence variation (SNP-free). Purple–<i>Acanthoxyla</i> reads mapped onto <i>Acanthoxyla</i> transcript assemblies; Pale green–<i>Clitarchus</i> reads mapped onto <i>Acanthoxyla</i> transcript assemblies; Pale purple–<i>Acanthoxyla</i> reads mapped onto <i>Clitarchus</i> transcript assemblies; Green–<i>Clitarchus</i> reads mapped onto <i>Clitarchus</i> transcript assemblies.</p

Alpha-actinin cDNA sequence diversity illustrates that allelic diversity of the stick insect <i>Acanthoxyla geisovii</i> is greater than within <i>Clitarchus hookeri</i> as expected of a hybrid.

<p>Within <i>Acanthoxyla geisovii</i> the SNP variant (allele) most similar to <i>Clitarchus hookeri</i> has approximately one third the expression level (inferred from read coverage) because this genome is triploid.</p

Results of general linear modelling of the regression between shell shape and water depth.

<p>Three models were tested: simple, regression independent of nominal taxonomic grouping; parallel, regression slope is independent of taxonomic grouping, but the intercept is not; non-parallel, slope and intercept depend on taxonomic grouping. The <i>p</i>-values on <i>F</i>-statistics indicate the probability that the simpler model in each comparison is adequate; significant <i>p</i>-values indicate that the more complex model is preferred (see text). In each comparison the more complex model is supported and, overall, the non-parallel model is preferred, indicating a correlation between depth and morphology.</p

Haplotype network (based on 573 bp of mtDNA <i>nad</i>2) of 27 <i>Alcithoe wilsonae</i> (open ovals) and 8 <i>Alcithoe knoxi</i> (filled ovals) demonstrates the lack of genetic structure related to morphology.

<p>Each haplotype differs by a single nucleotide, unsampled haplotypes are represented by black dots. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052083#pone-0052083-t001" target="_blank">Table 1</a> for sample codes.).</p

Results from AMOVA of <i>A. wilsonae</i> nad2 DNA sequneces, low Phi_PT values indicate high levels of genetic exchange between populations.

<p>Results from AMOVA of <i>A. wilsonae</i> nad2 DNA sequneces, low Phi_PT values indicate high levels of genetic exchange between populations.</p