Why facts matter: incomplete evidence and probabilistic induction triggered taxonomic inflation in Cathaica land snails from China (Eupulmonata, Camaenidae).

The Southeast Asian Pupillidae, commonly known as "micro snails", are prominent and ubiquitous members of limestone ecosystems particularly on the main land countries like Vietnam Cambodia, Laos, Myanmar, Malaysia and Thailand. They exhibit various peculiar biological features, including the unique shell morphology, the highly endemism. Their taxonomy was primarily developed by Pilsbry (1916-1918a, 1935), Steenberg (1925) and Baker (1935), based on shell and some anatomical characters. Although the generic level within this micro land snail family was lack of a modern phylogenetic perspective and the use of shell morphology and molecular characters for phylogenetic utility has not been established yet. This dissertation addresses this shortcoming by conducting phylogenetic studies on the Pupillidae, focusing mainly on the subfamily Gastrocoptinae, a major subgroup of the family in Southeast Asian region. Chapter 1, 2 review the current understanding of pupillid taxonomy, classification and their unique shell morphological characters. All of materials and methods are defied in Chapter 3. Using morphological characters, Chapter 4 studies on morphometric analysis and tests phylogenetic relationships among Thai pupillid genera and, as a result, Gyliotrachela and Hypselostoma do not forming the distinctive clusters by SWA character analysis and a monophyly of each genus is not supported. However, using the shell morphology alone may contain more homoplastic and increase the risk that the similarity of the character observed is not the result of the common ancestry but rather of parallel or convergent evolution. The current taxonomy of the Pupillidae is also revised based on combined phylogenetic analyses of two different gene sequence data, mitochondrial ribosomal 16S, and nuclear ribosomal 28S (Chapter 5). Although characterized by high levels of genetic differentiation and homoplasy, the molecular dataset provided a number of novel insights into gastrocoptine evolution and systematics. Nominal conspecifics of three genera with replicate samples (Gyliotrachela, Hypselostoma and Anauchen) occupied contiguous sections of treespace, however all three were paraphyletic. Two inferred examples of reductive loss in apertural lamellae were encountered: Aulacospira smaesarnensis, was firmly nested within an otherwise exclusively Gyliotrachela tip clade; the leaf-litter-dwelling Hypselostoma panhai, exhibited striking conchological differentiation from its geographically proximate rock-dwelling sister taxon H. erawan. The results caution against the unquestioned use of apertural dentition characteristics as diagnostic generic characters, imply that ecological transitions can lead to rapid morphological change, and suggest that a comprehensive sampling of both rock and leaf-litter lineages is required to fully flesh out phylogenetic relationships among regional pupillid microsnails. This dissertation also emphasize the utility of geographically proximate gastrocoptine taxa to establishing sister relationships for locally endemic species, irrespective of apparent morphological similarity. However, not all Thai gastrocoptines have localized ranges; Krobylos maehongsonensis, has apparently experienced geographically extensive patterns of gene flow and colonization. The results of these evolutionary studies not only increase our understanding of the phylogeny of the Pupillidae, but also provide the way to conduct the precise systematic classification, an important in biological basic study.

Genetic diversity of 39 Abelmoschus spp accessions was investigated at the DNA level with the random amplified polymorphic DNA (RAPD) procedure and at the phenotypic level with stable and highly heritable morphological characters. Thirty-one random decamer primers were used to amplify DNA by the polymerase chain reaction (PCR) and 103 RAPD fragments were generated. Thirteen quantitative and 14 qualitative characteristics were measured on the genotypes in field experiments using randomized complete blocks design with four replications. Dendrograms were generated for genetic distance based on molecular data (RAPD) and average distance from the morphological data. Classification of all genotypes based on the two methods gave similar results. It was observed a correlation of 0.62 between molecular and morphological data. On the other hand, the comparison between molecular distance and descriptors generated only by quantitative data showed that the correlation increased to 0.88, demonstrating that the qualitative descriptors had little influence on the genotypic discrimination. The most significant correlation (0.91) was verified between morphological descriptors distance and distance generated by quantitative data. These results showed that the quantitative descriptors and the molecular approach have high potential in the okra germplasm morphoagronomic characterization. This experiment also demonstrated that the investigated genotypes grouping pattern was similar to multivariate analysis.

Reappraising adaptive radiation

This paper represents the second part of our revisionary studies on the rhytidid fauna of southern Africa. The species discussed belong to the taxon Nata s.l. in which we recognise two genus-level lineages, Nata s.s. and Natella with six (three new) and one species respectively. We update the species-level taxonomy extensively in the light of new molecular and morphological data, and provide a comprehensive revision of all species, including keys. Detailed comparative morphological observations are provided for the distal reproductive tract, pulmonary cavity, mantle edge, radula and suprapedal gland. In addition, we present a summary of biological and ecological data including information on habitat preferences, feeding, prey items and mating behaviour. Although the two genera are well circumscribed in terms of both internal anatomy and molecular data, shell morphology is highly conserved, and species discrimination using shell characters alone is difficult. We have discovered three undescribed species within Nata and there is evidence that further research of a phylogeographic nature may uncover additional cryptic diversity. The geographic distributions of the species are discussed in relation to regional vegetation patterns and, as with the larger cannibal snail radiation, the Albany Thicket Biome emerges as a focus of endemism. Observations on the conservation status of all species are provided.New species: Nata aequiplicata sp. nov., Nata erugata sp. nov. and Nata watsoni sp. nov. Revised status: Natalina caffrula Melvill & Ponsonby, 1898 is transferred to the genus Nata and thought to be a synonym of Nata dumeticola (Benson, 1851).

Shell morphology and phylogenetic origin of oysters

The shell morphological variation of the periwinkle Littoraria angulifera (Lamarck, 1822) was studied in tropical northeast Brazilian mangroves. This area was selected because mangroves in different stages of regeneration, and thus different tree heights can be found. We evaluated whether differences in solar radiation due to differences in tree height influenced the distribution and shell morphology of L. angulifera, and carried out an experiment to test if individuals collected from mangroves with different tree heights differed in their resistance to desiccation. We also analysed if there were differences in L. angulifera shell length and shape between tidal levels within a mangrove. Finally, we tested if increased habitat complexity due to the presence of oysters could influence L. angulifera shell length and shape in different-statured mangroves. We predicted that the oysters could reduce desiccation stress on periwinkles in small-statured mangroves but have no effect in taller ones. Shell length and shape varied among mangroves, although a large variation within mangroves was also recorded. Shell proportionality (shell length:width ratio) increased with shell length, and this relationship differed among mangroves; however, no differences were found in a subsequent year. Individuals from small-statured mangroves survived longer than those from taller mangroves in the desiccation experiment, with a weak correlation between shell proportionality and loss of mass. The presence of oysters had no apparent influence on shell morphology that could be correlated with reduced desiccation stress. The patterns found varied greatly both at small and large spatial scales, suggesting that future studies should evaluate phenotypic and genetic variation at the same time to properly understand variation in L. angulifera shell morphology.

Invasive species often exhibit either evolved or plastic adaptations in response to spatially varying environmental conditions. We investigated whether evolved or plastic adaptation was driving variation in shell morphology among invasive populations of the New Zealand mud snail (Potamopyrgus antipodarum) in the western United States. We found that invasive populations exhibit considerable shell shape variation and inhabit a variety of flow velocity habitats. We investigated the importance of evolution and plasticity by examining variation in shell morphological traits 1) between the parental and F1 generations for each population and 2) among populations of the first lab generation (F1) in a common garden, full-sib design using Canonical Variate Analyses (CVA). We compared the F1 generation to the parental lineages and found significant differences in overall shell shape indicating a plastic response. However, when examining differences among the F1 populations, we found that they maintained among-population shell shape differences, indicating a genetic response. The F1 generation exhibited a smaller shell morph more suited to the low-flow common garden environment within a single generation. Our results suggest that phenotypic plasticity in conjunction with evolution may be driving variation in shell morphology of this widespread invasive snail.

Aestivation (summer dormancy) is a long-term multiday torpor in response to hot and dry periods. It has been detected in many species of terrestrial and aquatic turtles; however, several ecological and evolutionary aspects of chelonian aestivation remain to be evaluated and understood. We conducted a comparative exploration of macroevolutionary trends in turtle aestivation and tested the potential correlation of shell morphology with the aestivation duration. We compiled a dataset of aestivation status, aestivation times, and measurements of shell morphology of 225 turtle species. We reconstructed ancestral states along a time-calibrated phylogeny and tested different evolutionary models on the presence/absence of the aestivation trait. We also performed phylogenetic comparative analysis to explore several shell morphological traits likely associated with the duration time in aestivation behavior. We found evidence of aestivation in 44% of the evaluated turtle species. Aestivation times were longer in Chelidae, Pelomedusidae, Geoemydidae, and Kinosternidae, and the shortest times in Emydidae and Testudinidae. Aestivation behavior is a derived trait evolved independently and several times in the pleurodires and cryptodires turtle groups. We found some evolutionary trends in different turtle families, Pelomedusidae and Kinosternidae showed considerable increases in the presence of the aestivation trait, while families such as Podocnemididae, Trionychidae and Chelydridae showed important deductions for the same trait. Our results for the association between shell morphology and aestivation duration in turtles were contrasting among families. Overall, it was the contribution of several and different morphological traits that allow a positive and significant association with the aestivation times.

Alvarez, B., Crisp, M.D., Driver, F., Hooper, J.N.A. & Van Soest, R.W.M. (2000). Phylogenetic relationships of the family Axinellidae (Porifera: Demospongiae) using morphological and molecular data. —Zoologica Scripta, 29, 169–198.Twenty‐seven species of marine sponges belonging to Axinellidae and related groups (Halichondriidae, Dictyonellidae, Agelasida) were selected to test the monophyly of Axinellidae and investigate their phylogenetic relationships using parsimony and maximum likelihood methods. Partial 28S rDNA sequences, including the D3 domain, and traditional morphological characters (mainly skeletal ones) were used independently to construct phylogenetic trees. Sequences were aligned using the appropriate model of secondary structure of the RNA and compared to that produced by the multiple sequence alignment program, ClustalW. The alignment using secondary structure constraints produced a better estimate of the phylogeny and was demonstrated to be an effective and objective method.Results of the cladistic analyses of the molecular and morphological data sets were not fully congruent; the morphological data suggest that Axinellidae is monophyletic, however, the molecular data suggest that it is nonmonophyletic. The single most‐parsimonious tree derived from the molecular data showed that species ofAxinella(exceptA. polypoides) are united in a clade that is more closely related to members of Agelasida than to other species of Axinellidae; the remaining members of Axinellidae form a monophyletic group that is closely related to the families Dictyonellidae and Halichondriidae. The consensus tree of 20 most‐parsimonious trees from the morphological analysis, on the other hand, showed that all the sampled species of Axinellidae belong to a monophyletic group which is closely related to the species of Dictyonellidae and Halichondriidae. Only two branches were identical in both cladograms, the one uniting the species ofPtilocaulisandReniochalinaand the one with the species of Dictyonellidae.The robustness of the molecular and morphological trees (or parts of the trees), was tested using bootstrap, jack‐knife, PTP and T‐PTP tests. The results of the PTP test were significant indicating significant cladistic structure in both data sets. The bootstrap and jack‐knife values indicate that the molecular tree is in general better supported than the morphological one. The lack of morphological characters and the homoplastic nature of some may explain the weak support of the morphological tree. A T‐PTP test of nonmonophyly showed that the nonmonophyly of Axinellidae, as indicated by the results of the molecular analysis, is not significant; however, a T‐PTP test of monophyly of Axinellidae, as indicated by the morphological tree, produced significant results. This indicates that the monophyly of Axinellidae based on morphological data cannot be rejected; the family however, cannot be defined in terms of a unique diagnostic character common to all members of the ingroup.Tests of heterogeneity (reciprocal T‐PTP and partition homogeneity test) indicated that the data partitions are heterogeneous, which could be due to sampling errors (in either data set) or differences in the underlying phylogenies; therefore data were not combined in a single analysis. Further, both data sets are unequally sized (95 informative molecular characters vs. 16 informative morphological characters), which means that the molecular signal could swamp the morphological signal if the data is combined.Nonmonophyly of Axinellidae is supported by chemical and genetic evidence available in the literature and DNA sequences data of axinellid species from New Zealand. However, this needs to be confirmed using independent evidence from different genes (or gene regions), biochemistry, histology or cell ultrastructure. Therefore, no changes to the taxonomic position of the family in the higher classification are proposed at this stage.

The division Ascomycota(Fungi) contains a large number of taxa known to reproduce only asexually by the formation of conidia or other non-motile propagules produced by mitotic cellular devisions. They are called anamorphic, mitosporic, asexual or conidial fungi and ecologically, they are often found associated with plant debris in different stages of decay. In general, saprobic anamorphs of ascomycetous affinities are poorly studied and their outstanding diversity is currently underexplored. Phylogenetic relationships are unknown for many of them and they are still largely underrepresented in the current phylogenetic classification system of Fungi, with many morphologically defined anamorphic taxa still awaiting taxonomic reassessment in the light of molecular approaches. The increasing usage of molecular markers combined with robust statistical methods has allowed their phylogenetic affinities to be revealed and to gradually incorporate many of them into the different taxonomic groups of the division Ascomycota. However, the phylogenetic placement and taxonomic status of a large number of saprobic taxa remain unresolved due to the lack of DNA sequence data. The present dissertation aims to explore the rich but understudied diversity of those anamorphic fungi traditionally known as hyphomycetes that inhabit dead plant debris. It consists of five publications in which a polyphasic approach integrating morphological, developmental, cultural and molecular data was used to incorporate novel or incertae sedis taxa within Ascomycota and to make more sound decisions regarding their taxonomic status. Specific objectives include: 1. the collection, isolation and morphological characterization of selected anamorphic fungi representing putative new or interesting taxa of uncertain phylogenetic placement; 2. the generation of novel DNA sequence data to infer their phylogenetic relationships and to resolve their taxonomic affinities within Ascomycota; 3. the testing of any previously available morphologically based hypotheses on their putative position, generic placement or relationships with teleomorphic, pleomorphic or other anamorphic taxa; and 4. the determination of their generic validity, monophyly and taxonomic boundaries using molecular data and phylogenetic analyses methods. Materials studied in these five projects consisted of specimens collected during field work carried out by the author or collaborators in different countries including USA, the Czech Republic and Panama between the years 2014 and 2017. The target substrates were dead leaves of different palm trees, dead wood and bark of pines and twigs or stems of unknown shrubs and woody vines that are all known to harbor a rich saprobic mycobiota. Putative novelties or anamorphic taxa with unknown or poorly studied phylogenetic affinities were selected for further morphological and molecular investigation. Micromorphological studies were based on fungal structures observed on natural substrate, herbarium specimens and in culture. DNA was extracted from cultures and PCR amplification followed by Sanger sequencing was carried out using relevant molecular markers employed in fungal phylogenetic studies. Newly obtained DNA sequence data were analyzed following a standard phylogenetic analysis pipeline and phylogenetic relationships were reconstructed using character-based methods such as Maximum Likelihood and Bayesian inference. Conclusion is that anamorphic Ascomycota inhabiting dead plant debris represents a largely untapped source of biodiversity and information still in need of further exploration. A new capnodiaceous genus Castanedospora, seven new species named Taeniolella sabalicola, Hermatomyces bifurcatus, H. constrictus, H. megasporus, H. sphaericoides, H. verrucosus and Septonema lohmanii, and two new combinations, Castanedospora pachyanthicola and H. reticulatus, are proposed based on morphological and DNA sequence data. Molecular phylogenetics was confirmed as the tool of choice for the inference of relationships in novel or incertae sedis anamorphic fungi that are otherwise difficult to assess in the absence of a teleomorphic state. They were first resolved or revisited for several saprobic species such as Ernakulamia cochinensis, H. sphaericus, H. tucumanensis or Septonema fasciculare in a suitable framework for phylogenetic hypothesis testing. Molecular data allowed to fully incorporate all these taxa in Ascomycota, particularly within the classes Dothideomycetes and Sordariomycetes, and to provide a foundation for better taxonomic decisions on their classification. Large and polyphyletic genera such as Taeniolella, Sporidesmium and Septonema, partially treated in this work and containing mostly saprobic species of obscure affinities, remained in need of further investigation.

Leptopoma is a species rich genus with approximately 100 species documented. Species-level identification in this group has been based on shell morphology and colouration, as well as some anatomical features based on small sample sizes. However, the implications of the inter- and intra-species variations in shell form to the taxonomy of Leptopoma species and the congruency of its current shell based taxonomy with its molecular phylogeny are still unclear. There are four Leptopoma species found in Sabah, Borneo, and their taxonomy status remains uncertain due to substantial variation in shell forms. This study focuses on the phylogenetic relationships and geographical variation in shell form of three Leptopoma species from Sabah. The phylogenetic relationship of these species was first estimated by performing Maximum Likelihood and Bayesian analysis based on mitochondrial genes (16S rDNA and COI) and nuclear gene (ITS-1). Then, a total of six quantitative shell characters (i.e., shell height, shell width, aperture height, aperture width, shell spire height, and ratio of shell height to width) and three qualitative shell characters (i.e., shell colour patterns, spiral ridges, and dark apertural band) of the specimens were mapped across the phylogenetic tree and tested for phylogenetic signals. Data on shell characters of Leptopoma sericatum and Leptopoma pellucidum from two different locations (i.e., Balambangan Island and Kinabatangan) where both species occurred sympatrically were then obtained to examine the geographical variations in shell form. The molecular phylogenetic analyses suggested that each of the three Leptopoma species was monophyletic and indicated congruence with only one of the shell characters (i.e., shell spiral ridges) in the current morphological-based classification. Although the geographical variation analyses suggested some of the shell characters indicating inter-species differences between the two Leptopoma species, these also pointed to intra-species differences between populations from different locations. This study on Leptopoma species is based on small sample size and the findings appear only applicable to Leptopoma species in Sabah. Nevertheless, we anticipate this study to be a starting point for more detailed investigations to include the other still little-known (ca. 100) Leptopoma species and highlights a need to assess variations in shell characters before they could be used in species classification.

The land snail genus Rumina shows much intra- and interspecific variation in shell morphology. Three morphospecies are commonly recognized: R. decollata, R. saharica and R. paivae. The descriptions of these three morphospecies were based on differences in shell and genital characters for R. saharica and R. decollata, and shell size for R. paivae. However, recently DNA-sequence data have suggested that these morphospecies comprise at least seven phylogenetic species (R. decollata molecular operational taxonomic units (MOTUs) A –F and R. saharica). The present study explores to what extent these phylogenetic species are morphologically diagnosable and/or can be reconciled with the three currently recognized morphospecies. It shows that: (1) R. saharica, and to a lesser extent R. decollata MOTUs A and Eb, are significantly differentiated from the other phylogenetic species by both genital and shell characters, and can be regarded as three diagnosable biological and phylogenetic species; (2) there are no diagnostic genital features for R. paivae, so that this taxon should not be treated as a separate species; (3) none of the other DNA-based R. decollata MOTUs could be consistently differentiated by the shell and genital characters analysed in this study, so that their status needs further scrutiny.

Many molluscan species exhibit a high degree of shell morphological plasticity in their shape (including sculptures), size and colour patterns, which can vary significantly depending on environmental conditions. These shell morphological variations make it challenging to differentiate species, based on morphology alone, often resulting in various taxonomic errors, such as misidentifications, overlooking cryptic species diversity or a plethora of nominal species. The genus Siphonaria constitutes a significant component of the macrobenthic invertebrate fauna in intertidal habitats across temperate to tropical regions. Given the limited attention to shell variation in previous taxonomic studies on the Korean Siphonaria species, the extensive range of ecophenotypic shell variations documented in this group raises questions about the taxonomic validity of previously reported Siphonaria species in Korea. The present study provides a comprehensive taxonomic review of Korean Siphonaria species using a combination of shell morphology, radula structure and phylogenetic analysis of the mtDNA cox1 sequences. This integrative analysis confirmed the validity of S.acmaeoides, S.japonica and S.sirius in Korea, highlighting differences in shell and siphonal groove morphology amongst these species. Detailed descriptions of shell and radula characteristics, along with mtDNA cox1 sequences as DNA barcodes, are also provided, which are very useful for the accurate identification of Siphonaria species. Unlike these three Siphonaria species, the taxonomic validity of the four other species (S.coreensis, S.javanica, S.laciniosa and S.rucuana) previously reported from Korean waters is questionable, given their documented geographic distribution ranges and the potential misidentification of shell variants in Korean malacofaunal studies.

Taxonomic identification of alien bivalve species in the Mediterranean Sea can be challenging because of high morphological variability and the occurrence of cryptic species complexes, as in the case of pearl oysters. While the presence of the Lessepsian species Pinctada radiata is well-established in the eastern Mediterranean Sea, the presence of P. fucata was recently suggested based on morphological data. In this study we performed an integrative assessment based on morphological and genetic data on pearl oysters collected across the Mediterranean Basin. Molecular species delimitation and phylogenetic analyses allowed a straightforward taxonomic assignment of all collected specimens to P. radiata. These specimens show the entire suite of morphological character states considered diagnostic of either P. fucata or P. radiata by previous studies. This finding clearly demonstrates that these morphological characters have no taxonomic value, and their variability observed in specimens from the Mediterranean Sea represents (part of) the intraspecific variability of P. radiata. While no evidence has been found for the presence of P. fucata, the earlier occurrences of P. radiata from the eastern and southern Mediterranean Sea are complemented with verified occurrence in the western and northern regions, demonstrating a further spreading of this non-native species throughout the Mediterranean Sea. This study clarifies the taxonomic identification and geographical distribution of pearl oysters in the Mediterranean Sea and substantiates the importance of molecular identification of alien bivalves characterized by extensive variation in shell characters.

Increases in human activity and climate change are factors that are expected to augment the number and impact of alien species in the northern regions. In the present paper, we report the discovery of a population of Asian Corbicula clams in the North of Russia near the Arctic frontier (64°N, 40°E), which is the most northern record of these invaders in the world. This population contains two distinct lineages that are living in sympatry in a man-made channel of the Arkhangelsk thermal power plant. The first lineage belongs to the morphotype Rlc by shell morphology but reveals the FW5 mtDNA haplotype (COI and 16S rRNA genes), which was typically recorded in the morphotype R. The second lineage reveals the shell morphological pattern of the morphotype R but has the FW17 mtDNA haplotype, which usually occurs in specimens of the morphotype S. Both forms that were identified, based on conchological and anatomical features and differences in mtDNA haplotypes, reveal the same 28S rDNA sequences but show intragenomic nDNA variability with two different variants of the 28S rDNA gene within each individual (allelic sequence heterogeneity). The discrepancy between mtDNA data and shell morphology could be explained by the well-known mechanism that is associated with androgenesis together with “egg parasitism” (mitochondrial genome-capture) in invasive Corbicula clams. The intragenomic 28S rDNA variability may be due to a hybridization event which can join different alleles from the two diploid (or polyploid) parental taxa to an allopolyploid descendant. We found that the lineages that rarely occur in European populations are exclusively established in a population near the Arctic frontier. Moreover, specimens belonging to the R-morphotype with the FW17 mtDNA haplotype that has sporadic occurrences in central Europe is prevalent (> 90% of a total sample) in a mollusk assemblage at the studied site in northern Russia. Additionally, we show that the life span of Corbicula clams in high latitudes may be longer than 1 year. Isolated Corbicula populations in artificially heated or geothermal reservoirs may therefore be primary sources for a broad invasion to seasonal waterbodies of the high-latitude regions in the future as climate warming progresses.