Morphology-based Taxonomy Research Articles

From 20 to 2? Landmark-based geometric morphometrics reveal negligible wing-shape divergence between 20 subspecies of the Apollo butterfly, Parnassius apollo (Lepidoptera, Papilionidae), in the Carpatho-Pannonian region

For effective conservation management of endangered taxa, it is important to define operational units for conservation. In the absence of detailed genetic analyses, morphology-based taxonomy is often used as a surrogate. The Apollo butterfly, Parnassius apollo, is one of the most endangered butterfly species in Europe (considered as a flagship species) with 26 subspecies rank taxa described from the Carpatho-Pannonian region (Central Europe), often based on old, one-by-one descriptions. We applied landmark-based geometric morphometrics on wing shape to determine the number of morphologically distinguishable groups in the region, based on 949 males and 477 females from 20 Carpatho-Pannonian putative subspecies (both extant and potentially extinct). We found a single division between the Eastern Carpathian populations (described as two subspecies: ssp. transsylvanicus and ssp. rosenius) and the rest of the populations (including our outgroup from the Swiss Alps). Since P. apollo was not observed in the Eastern Carpathians in the last two decades, and the currently known extant populations in the Carpatho-Pannonian region are all located in the Northern Carpathians, our results support a single conservation unit in the region. We suggest that (i) extensive monitoring is needed to reveal whether the unique Eastern Carpathian populations have really gone extinct and (ii) more taxonomical/phylogenetic studies on Central European P. apollo are needed for establishing the taxonomy of the species and efficient conservation strategies. We emphasize that modern integrative taxonomy is not only important for clarifying taxonomical issues, but also for providing basis for sound conservation management.

A new species of coral-dwelling crab (Decapoda: Brachyura: Cryptochiridae: Opecarcinus) from the West Pacific

Based on material acquired from Green Island, Taiwan, using a combined approach of traditional morphology-based taxonomy and molecular barcoding, we describe a new species of coral-dwelling crab, Opecarcinus ngankeeae sp. nov., from the scleractinian hosts Pavona decussata and P. varians (family Agariciidae). The DNA sequences of the present species matched with O. sp. SET6, associated with plate-forming Leptoseris and Pavona corals, available on Genbank, provided by Xu et al. (2022). The geographical distribution of O. ngankeeae sp. nov. spans from the Coral Triangle and Taiwan to Japan in West Pacific.

Diversity of Cellular Slime Molds (Dictyostelids) in the Fanjing Mountain Nature Reserve and Geographical Distribution Comparisons with Other Representative Nature Reserves in Different Climate Zones of China.

Protected areas are widely considered an essential strategy for biodiversity conservation. Dictyostelids are unique protists known to have important ecological functions in promoting soil and plant health through their top-down regulation of ecosystem processes, such as decomposition, that involve bacterial populations. But the relationship between dictyostelid diversity within protected areas remains poorly understood, especially on a large scale. Herein, we report data on the distribution of dictyostelids, identified with ITS + SSU rRNA molecular and morphology-based taxonomy, from soil samples collected in the Fanjing Mountain protected area of Guizhou Province, Southwest China. We compared the biodiversity data of dictyostelids in Fanjing Mountain with similar data from previously sampled sites in four other protected areas, including Changbai Mountain (CB), Gushan Mountain (GS), Baiyun Mountain (BY), and Qinghai-Tibet Plateau (QT) in China. We identified four species of dictyostelids belonging to three genera (Dictyostelium, Heterostelium, and Polysphondylium) and herein provide information on the taxonomy of these species. Two species (Heterostelium pallidum and Dictyostelium purpureum) are common and widely distributed throughout the world, but one species (Polysphondylium fuscans) was new to China. Our data indicate that there is no distinguishable significant correlation between the dictyostelid species studied and environmental factors. Overall, the similarity index between Baiyun Mountain in Henan Province and Fanjing Mountain in Guizhou Province, located at approximately the same longitude, is the highest, and the Jaccard similarity coefficients (Jaccard index) of family, genus, and species are 100%, 100%, and 12.5%, respectively. From a species perspective, species in the same climate zone are not closely related, but obvious geographical distributions are evident in different climate zones. This preliminary study provided evidence of the ecological adaptation of dictyostelids to different biological niches.

Performance evaluation of Moringa oleifera seeds aqueous extract for removing Microcystis aeruginosa and microcystins from municipal treated-water.

Introduction: Toxic microcystins (MCs) produced by cyanoprokaryotes -particularly by the cosmopolitan cyanobacterium Microcystis aeruginosa- pose adverse effects on aquatic organisms and their ecosystem and may also cause serious impacts on human health. These harmful monocyclic heptapeptides are the most prevalent cyanotoxins reported in freshwaters and must be eliminated for avoiding MCs release in receiving water bodies. Hence, this work aimed to test the efficacy of Moringa oleifera seeds water-based extract (MO) as a natural coagulant for removing cyanobacteria (especially M. aeruginosa), microalgae, and its associated MCs from pre-treated municipal wastewaters. Methodology: Four different MO coagulant doses (25, 50, 75 and 100mgL-1) were investigated for cyanobacteria and microalgae removal by conventional coagulation assays and morphology-based taxonomy studies. Additionally, water turbidity and chlorophyll a (Chl a) content were also determined. Further, the presence and concentration of MCs soluble in water, remaining in the particulate fraction, and flocculated within the residual sludge were assessed using high-performance liquid chromatography coupled with diode array detection (HPLC-DAD). Results: The treatment with MO at 100mg L-1 substantially reduced the number of cyanobacterial and microalgal species in the treated samples (average removal rate of 93.8% and 86.9%, respectively). These results agreed with a ∼44% concomitant reduction in Chl a and ∼97% reduction in water turbidity (a surrogate marker for suspended solids content). Notably, MCs concentrations in the treated water were significantly lowered to 0.6 ± 0.1µg L-1 after addition of 100mg L-1 MO. This value is below the WHO recommended limits for MCs presence in drinking water (<1.0µg L-1). Discussion: The present study provides promising insights into the applicability of MO as a cost-effective, reliable, and sustainable natural coagulant, particularly for using in developing countries, to eliminate harmful cyanobacteria and cyanotoxins in municipal water treatment facilities.

Identification and diversity assessment of Ziziphus species using DNA barcoding

The genus Ziziphus, belonging to the family Rhamnaceae, is of great economic and medicinal importance worldwide due to the value of its fruits in household income and food security. However, many species within this genus are phenotypically identical, leading to imprecise identification and hindering their conservation, variation in the gene pool, and exploitation. To address this issue, we collected 26 individuals from different regions of Khyber Pakhtunkhwa, Pakistan and Shanxi province, China, to assess the efficiency of DNA barcoding regions in identifying Ziziphus species. We tested the ability of five DNA barcodes regions, namely ITS, matK, rbcL, trnH-psbA, and trnL-UAA, to distinguish between Ziziphus species as an alternative approach for species identification and the phylogenetic reconstruction. The results of the study, as the success rate of PCR amplification of matK, trnL-UAA, and ITS, provided the highest species resolution with 100 % accuracy, except for trnH-psbA (90.78 %). ITS and matK were identified as the most effective single barcode, with a species identification success rate of 87.4 %±0.98 % and 84.89 %±0.66 %. These results suggest that matK, trnL-UAA, and ITS are universal barcode regions for identification of all Ziziphus species identification and their discrimination power to differentiate. In contrast, the combination of trnL-UAA+ITS region displayed the highest species identification rate (92.1 %), the five barcodes region combination, and superior to any barcodes regions combination that included rbcL+trnH-psbA. Additionally, the study found high haplotypes and nucleotide diversity in Pakistani Ziziphus species, mainly in the ITS barcode, with values of 0.84 and 0.0086, respectively. In conclusion, the study establishes that the DNA fragments ITS, trnL-UAA, and matK are effective in identifying and distinguishing Ziziphus species. The approach can be considered as an accurate tool for identifying these species and will be used as a universal barcode's region for Ziziphus species. DNA barcoding is a useful approach and provides a reliable and effective means for Ziziphus discrimination, and in combination with morphology-based taxonomy will be a robust approach for tackling taxonomically complex species. Additionally, this study provides valuable insights into the genomic architecture of Ziziphus species, which has implications for their classification, conservation, and germplasm resource improvement. This research provides a novel approach to the problem of incorrectly identifying economically and medicinally important species, particularly those in the genus Ziziphus.

Marine Flora of French Polynesia: An Updated List Using DNA Barcoding and Traditional Approaches.

Located in the heart of the South Pacific Ocean, the French Polynesian islands represent a remarkable setting for biological colonization and diversification, because of their isolation. Our knowledge of this region's biodiversity is nevertheless still incomplete for many groups of organisms. In the late 1990s and 2000s, a series of publications provided the first checklists of French Polynesian marine algae, including the Chlorophyta, Rhodophyta, Ochrophyta, and Cyanobacteria, established mostly on traditional morphology-based taxonomy. We initiated a project to systematically DNA barcode the marine flora of French Polynesia. Based on a large collection of ~2452 specimens, made between 2014 and 2023, across the five French Polynesian archipelagos, we re-assessed the marine floral species diversity (Alismatales, Cyanobacteria, Rhodophyta, Ochrophyta, Chlorophyta) using DNA barcoding in concert with morphology-based classification. We provide here a major revision of French Polynesian marine flora, with an updated listing of 702 species including 119 Chlorophyta, 169 Cyanobacteria, 92 Ochrophyta, 320 Rhodophyta, and 2 seagrass species-nearly a two-fold increase from previous estimates. This study significantly improves our knowledge of French Polynesian marine diversity and provides a valuable DNA barcode reference library for identification purposes and future taxonomic and conservation studies. A significant part of the diversity uncovered from French Polynesia corresponds to unidentified lineages, which will require careful future taxonomic investigation.

Intra-island patterns of cryptic diversity within an oceanic island: Insights from the differentiation of Schizidium Verhoeff, 1901 (Oniscidea, Armadillidiidae) within Cyprus, with descriptions of two new species

Cyprus is a Mediterranean island of volcanic origin isolated for at least 5.3 Myr from surrounding continental areas. The present study focuses on the diversification of the isopod genus Schizidium within the island, including also specimens from surrounding continental areas. The genus Schizidium sensu lato is probably non monophyletic, comprising 26 species distributed from Greece to Iran. Up to date the only representative of the genus reported from Cyprus was Schizidium fissum. Aiming to investigate the patterns of genetic diversity within the focal island, to evaluate the morphology-based taxonomy of the species in the genus, and at the same time to explore phylogenetic relationships with mainland populations, we applied genome-wide ddRADseq as well as Sanger sequencing targeting three mitochondrial (16S, COI and 12S) and the nuclear NaK loci. Results of phylogenetic analyses support the existence of two distinct epigean Schizidium clades with well-defined geographic boundaries that conform to the known paleogeography of Cyprus, plus one endogean clade with restricted distribution within the island. Genetic data and morphology corroborate the assignment of this latter endogean clade to a new species, Schizidium myrrae n. sp. The two epigean clades are also considered as distinct species, one corresponding to the known S. fissum at the eastern part of the island (Pentadaktylos massif) and the other to the newly described S. christosi n. sp. distributed along the western part of the island (Troodos massif). Even though detailed examination of many specimens could not retrieve any morphological differences among representatives of these two clades, clado-chronological analysis indicates a long isolation between them, estimated at ∼ 9 Mya, as well as the sharing of a common ancestor with S. tiberianum from Israel at ∼ 15 Mya. Hence, we can consider these epigean Schizidium species as one more case of cryptic diversity on Cyprus, exhibiting similar patterns with the recently described case in the genus Armadillo.

The genus Acicula W. Hartmann, 1821 in the Caucasus (Gastropoda: Caenogastropoda: Aciculidae)

The Aciculidae is a family of terrestrial operculate snails with a western Palaearctic distribution. Historically three Acicula species were reported from the Caucasus Mountains, which represents the eastern edge of the family's distribution: A. limbata, A. moussoni and A. parcelineata, all from a few localities. We examined extensive material from the entire range of the Caucasus Mountains by means of classic morphology-based taxonomy and geometric morphometries. We arrived at the following conclusions: (1) At least three species inhabit the Caucasus Mountains, two of which are problematic taxonomically and regarding their nomenclature. (2) The two syntypes of A. moussoni have been lost, and both lectotype designations by the former authors were invalid. Therefore, we designate a neotype for A. moussoni. (3) Acicula limbata was described from Lower Miocene deposits of the Czech Republic. Although the fossil Central European and extant Caucasian shells differ only slightly from one another, it is highly unlikely that they would belong to the same species. Therefore, we suggest using A. limbata only for the Czech fossil species and A. moussoni for the Caucasian extant species. (4) In multiple samples, the generally smaller Acicula parcelineata was found syntopic with the generally larger A. moussoni. Therefore, the two are possibly distinct species. However, it is very difficult to distinguish, and the problem of A. moussoni and A. parcelineata needs further attention. (5) A species with a bullet (or torpedo)-shaped shell and an opisthocline apertural profile from the northern Caucasus region has been treated as A. moussoni by previous authors, but it is distinct from the other two Acicula species by consistent conchological characters and was also found with them in syntopy, and is described here as Acicula telum Páll-Gergely, n. sp.

Systematics of Ditaxinae and Related Lineages within the Subfamily Acalyphoideae (Euphorbiaceae) Based on Molecular Phylogenetics.

The subtribe Ditaxinae in the plant family Euphorbiaceae is composed of five genera (Argythamnia, Caperonia, Chiropetalum, Ditaxis and Philyra) and approximately 120 species of perennial herbs (rarely annual) to treelets. The subtribe is distributed throughout the Americas, with the exception of Caperonia, which also occurs in tropical Africa and Madagascar. Under the current classification, Ditaxinae includes genera with a questionable morphology-based taxonomy, especially Argythamnia, Chiropetalum and Ditaxis. Moreover, phylogenetic relationships among genera are largely unexplored, with previous works sampling <10% of taxa, showing Ditaxinae as paraphyletic. In this study, we inferred the phylogenetic relationships within Ditaxinae and related taxa using a dataset of nuclear (ETS, ITS) and plastid (petD, trnLF, trnTL) DNA sequences and a wide taxon sampling (60%). We confirmed the paraphyly of Ditaxinae and Ditaxis, both with high support. Following our phylogenetic results, we combined Ditaxis in Argythamnia and upgraded Ditaxinae to the tribe level (Ditaxeae). We also established and described the tribe Caperonieae based on Caperonia, and transferred Philyra to the tribe Adelieae, along with Adelia, Garciadelia, Lasiocroton and Leucocroton. Finally, we discuss the main morphological synapomorphies for the genera and tribes and provide a taxonomic treatment, including all species recognized under each genus.

Genetic-morphological uncoupling and crypsis in Ectinogonia (Coleoptera: Buprestidae) suggest a complex evolutionary history in these polymorphic jewel beetles from Chile

Abstract Ectinogonia is a genus of jewel beetles from the western slope of the Andes Cordillera, inhabiting arid and semiarid ecosystems in different biogeographical provinces. Most of the species in this genus have undergone several rearrangements and misassignments over time, making the taxonomic history of Ectinogonia particularly complex. Recent studies based on molecular phylogenies suggest that the taxonomy of the genus should be revised using genetic tools. Because some species are polymorphic and others cryptic and monomorphic, species diversity may be over- or underestimated. We performed here the first comprehensive molecular phylogeny for Ectinogonia s.s. using nuclear and mitochondrial genes. We also performed species delimitation analyses to estimate diversity within Ectinogonia. Results show that Ectinogonia is divided into two main groups: the artificial ‘southern group’ and the natural ‘northern clade’. The southern group matched with the current morphology-based taxonomy, whereas the northern clade contained the largest number of species, several of which do not fit with the current taxonomy. There was both genetic-morphological uncoupling and crypsis by convergence, suggesting a complex evolutionary history. We also provide a new taxonomic arrangement based on our results.

Size does not matter: molecular phylogeny reveals one of the largest trematodes from vertebrates, the enigmatic Ithyoclinostomum dimorphum, as a species of Clinostomum (Trematoda: Clinostomidae).

Despite the recent advances raised in the molecular era to the taxonomic knowledge of species of the family Clinostomidae, especially those belonging to the specious genus Clinostomum, some groups of these vertebrate parasites remain poorly studied. This is the case of species of the enigmatic genus Ithyoclinostomum Witenberg, 1926, until recently monotypic and restricted to South America, but with its occurrence expanded to North America after the description of I. yamagutii Rosser et al., 2020. Nevertheless, molecular data for the type species of the genus, Ithyoclinostomum dimorphum (Diesing, 1850), is lacking so far. In the present study, large clinostomid metacercariae morphologically indistinguishable from I. dimorphum were obtained from two erythrinid fishes from the Rio Doce River, Southeast Brazil. Samples of the parasites were subjected to a multigene (28S rDNA, ITS and cox1) molecular characterization followed by phylogenetic reconstructions. Phylogenies based on single-gene and concatenated datasets revealed unequivocally that I. dimorphum falls in a well-supported clade together with species of the genus Clinostomum. Moreover, the molecular divergences observed in relation to Clinostomum spp. [ranges of 2.4–6%, 2.4–3.8% and 14.7–19.3% for the ITS, 28S and cox1 genes, respectively] are compatible with a congeneric status with these species. Therefore, the genus Ithyoclinostomum is here synonymized with Clinostomum Leidy, 1856 and C. dimorphum (Diesing, 1850) Braun, 1899 re-established. In the phylogenetic analysis, the recently described 'Ithyoclinostomum' yamagutii, presented as an isolated, independent lineage, showing significant molecular divergences to C. dimorphum (12.6%, 7.6%, 18,6% for the ITS, 28S and cox1 genes, respectively). However, given the complex scenario raised in the morphology-based taxonomy of Clinostomidae, we took a conservative approach by not proposing a new genus to 'I.' yamagutii until molecular data of other clinostomid genus from birds, Clinostomatopsis, become available. Data here presented reveals that body size is not a useful criterion for higher-level classification in Clinostomidae. Finally, we highlighted the importance of the availability of molecular data for the type species of trematode genera proposed from South America to support a trans- or intercontinental distribution.

New Insights Into the Backbone Phylogeny and Character Evolution of Corydalis (Papaveraceae) Based on Plastome Data.

A robust backbone phylogeny is fundamental for developing a stable classification and is instructive for further research. However, it was still not available for Corydalis DC., a species-rich (> 500 species), ecologically and medically important, but taxonomically notoriously difficult genus. Here, we constructed backbone phylogeny and estimated the divergence of Corydalis based on the plastome data from 39 Corydalis species (32 newly sequenced), which represent ca. 80% of sections and series across this genus. Our phylogenetic analyses recovered six fully supported main clades (I–VI) and provided full support for the majority of lineages within Corydalis. Section Archaeocapnos was unexpectedly turned out to be sister to the rest of the subg. Corydalis s. l. (clades IV–VI), thus treating as a distinct clade (clade III) to render all the main clades monophyletic. Additionally, some unusual plastome structural rearrangements were constantly detected within Corydalis and were proven to be lineage-specific in this study, which, in turn, provided further support to our phylogeny. A segment containing five genes (trnV-UAC–rbcL) in the plastome's LSC region was either normally located downstream of the ndhC gene in clade I species or translocated downstream of the atpH gene in clade II species or translocated to downstream of the trnK-UUU gene in clade III–VI species. The unique large inversion (ca. 50 kb) in the plastome LSC region of clade III species, representing an intermediate stage of the above translocation in clades IV–VI, firmly supported clade III as a distinct and early diverged clade within this large lineage (clades III–VI). Our phylogeny contradicted substantially with the morphology-based taxonomy, rejected the treatment of tuberous species as an independent evolutionary group, and proved that some commonly used diagnostic characters (e.g., root and rhizome) were results of convergent evolution, suggestive of unreliability in Corydalis. We dated the origin of crown Corydalis to the early Eocene (crown age 49.08 Ma) and revealed possible explosive radiation around 25 Ma, coinciding with the drastic uplift of the Qinghai-Tibetan Plateau in Oligocene and Miocene. This study provided the most reliable and robust backbone phylogeny of Corydalis to date and shed some new insights on the evolution of Corydalis.

The &lt;i&gt;COI&lt;/i&gt; haplotype diversity of the pelagic polychaete &lt;i&gt;Tomopteris&lt;/i&gt; (Annelida: Tomopteridae) collected from the Pacific coast off Kii Peninsula, central Japan

The polychaete genus Tomopteris is an important component of the zooplankton and is also known for its communication using bioluminescence. However, there have been few studies about the species diversity and ecology of Tomopteris because of taxonomic and handling difficulties resulting from the fragility of their bodies. In this study, we performed DNA barcoding and species delimitation analysis to better understand the species diversity of Tomopteris using 17 specimens collected on a research cruise conducted off the eastern Kii Peninsula, Japan. Due to damage to the detailed morphological characteristics necessary for identifications based on the current taxonomy, none of the specimens was morphologically identifiable at the species level. The analyses of the mitochondrial cytochrome c oxidase subunit I (COI) and nuclear large ribosomal subunit (28S) RNA gene sequences revealed 7–8 molecularly delimited species among them, which was greater than the number of species currently recognized in Japan. These results suggest that the traditional morphology-based taxonomy overlooks the taxonomic complexity of Tomopteris, thus a combinatory analysis of both morphology and DNA barcoding will be desirable for assessing the true species biodiversity of Tomopteris.

Taxonomy of the weed species of the genus Echinochloa (Poaceae, Paniceae) in Southwestern Europe: Exploring the confused current state of affairs.

The taxonomy of Echinochloa, a predominantly tropical to warm-temperate genus of 40-50 species, including some of the world's worst weeds, is still poorly understood. This is because some species, including the extremely widespread E.crus-galli, show a wide range of morphological, physiological and ecological variation, in part the result of a complex recent evolutionary history. Furthermore, there is often a dearth of clear distinguishing features among species. The same applies to the species established in Southwestern Europe, where unintentionally introduced populations have now established themselves as important weeds of crops, especially maize and rice. Taxonomic and nomenclatural confusion hampers progress in weed science. In this study, we give an identification key that covers the weedy taxa encountered in Southwestern Europe, followed by notes on taxonomy and nomenclature. Moreover, a lectotype is designated for Echinochloafrumentacea. It is argued that current confusion cannot be overcome without including populations of Eastern Asian origin in taxonomic studies and without the joint efforts of experts in the fields of weed science, morphology-based taxonomy, genomics and phylogenetics.

Genome Skimming Contributes to Clarifying Species Limits in Paris Section Axiparis (Melanthiaceae).

Paris L. section Axiparis H. Li (Melanthiaceae) is a taxonomically perplexing taxon with considerable confusion regarding species delimitation. Based on the analyses of morphology and geographic distribution of each species currently recognized in the taxon, we propose a revision scheme that reduces the number of species in P. sect. Axiparis from nine to two. To verify this taxonomic proposal, we employed a genome skimming approach to recover the plastid genomes (plastomes) and nuclear ribosomal DNA (nrDNA) regions of 51 individual plants across the nine described species of P. sect. Axiparis by sampling multiple accessions per species. The species boundaries within P. sect. Axiparis were explored using phylogenetic inference and three different sequence-based species delimitation methods (ABGD, mPTP, and SDP). The mutually reinforcing results indicate that there are two species-level taxonomic units in P. sect. Axiparis (Paris forrestii s.l. and P. vaniotii s.l.) that exhibit morphological uniqueness, non-overlapping distribution, genetic distinctiveness, and potential reproductive isolation, providing strong support to the proposed species delimitation scheme. This study confirms that previous morphology-based taxonomy overemphasized intraspecific and minor morphological differences to delineate species boundaries, therefore resulting in an overestimation of the true species diversity of P. sect. Axiparis. The findings clarify species limits and will facilitate robust taxonomic revision in P. sect. Axiparis.

The Complete Chloroplast Genomes of Primula obconica Provide Insight That Neither Species nor Natural Section Represent Monophyletic Taxa in Primula (Primulaceae).

The genus Primula (Primulaceae) comprises more than 500 species, with 300 species distributed in China. The contradictory results between systematic analyses and morphology-based taxonomy make taxonomy studies difficult. Furthermore, frequent introgression between closely related species of Primula can result in non-monophyletic species. In this study, the complete chloroplast genome of sixteen Primula obconica subsp. obconica individuals were assembled and compared with 84 accessions of 74 species from 21 sections of the 24 sections of the genus in China. The plastome sizes of P. obconica subsp. obconica range from 153,584 bp to 154,028 bp. Genome-wide variations were detected, and 1915 high-quality SNPs and 346 InDels were found. Most SNPs were detected in downstream and upstream gene regions (45.549% and 41.91%). Two cultivated accessions, ZP1 and ZP2, were abundant with SSRs. Moreover, 12 SSRs shared by 9 accessions showed variations that may be used as molecular markers for population genetic studies. The phylogenetic tree showed that P. obconica subsp. obconica cluster into two independent clades. Two subspecies have highly recognizable morphological characteristics, isolated geographical distribution areas, and distinct phylogenetic relationships compared with P. obconica subsp. obconica. We elevate the two subspecies of P. obconica to separate species. Our phylogenetic tree is largely inconsistent with morphology-based taxonomy. Twenty-one sections of Primula were mainly divided into three clades. The monophyly of Sect. Auganthus, Sect. Minutissimae, Sect. Sikkimensis, Sect. Petiolares, and Sect. Ranunculoides are well supported in the phylogenetic tree. The Sect. Obconicolisteri, Sect. Monocarpicae, Sect. Carolinella, Sect. Cortusoides, Sect. Aleuritia, Sect. Denticulata, Sect. Proliferae Pax, and Sect. Crystallophlomis are not a monophyletic group. The possible explanations for non-monophyly may be hybridization, polyploidization, recent introgression, incorrect taxonomy, or chloroplast capture. Multiple genomic data and population genetic studies are therefore needed to reveal the evolutionary history of Primula. Our results provided valuable information for intraspecific variation and phylogenetic relationships within Primula.

In Silico Evaluation of the Taxonomic Resolution and Coverage of the COI Region and Alternative Barcode Markers for Echinoderms

DNA barcoding methods are potentially useful for the detection and identification of species difficult to identify using morphology-based taxonomy, such as the larvae of marine taxa. Combining barcoding methods with massively parallel sequencing and low-cost DNA library preparation improves the feasibility of processing several samples but limits the taxonomic resolution of resulting sequences. In light of these limitations, we looked for suitable barcode regions for echinoderm larvae, first by examining the cytochrome c oxidase subunit I (COI) region, the most commonly used barcode for echinoderms. Using a dataset containing over 4000 COI sequences representing nearly 400 echinoderm species across all five extant classes, the COI gene was found to lack a sufficiently conserved region for barcoding. Taxonomic resolution was also found lacking, with no clear barcode gap across classes and the results of Automatic Barcode Gap Discovery (ABGD) failing to reflect current taxonomic assignments. While sequences may be mislabeled, the aggregation of multiple species in one ABGD partition suggests that course-grained taxonomic resolution may still result from using COI alone. The search for short alternative mitochondrial regions across 110 genomes of distinct species using ecoPrimers software revealed that class-specific primers targeting 12S and 16S ribosomal RNA (rRNA) regions are prime candidates for barcoding echinoderm sequences. The results of in silico PCR across the 110 mitochondrial genomes indicate that compared to existing COI primers, the candidate 12S and 16S primers yield similar taxonomic coverage, with slightly lower resolution. Taking into consideration the previous analysis of COI, we suggest the use of multiple markers (COI, 12S, 16S) to adequately capture the understudied diversity of echinoderm larvae. In vitro PCR of the 12S and 16S primers is also needed to validate the results of the in silico analyses.

Morphological and molecular diversity patterns of the genus Tropodiaptomus Kiefer, 1932 (Copepoda, Calanoida, Diaptomidae) in Thailand

Tropodiaptomus is one of the most specious genera in the family Diaptomidae, but it is often rare in terms of distribution and abundance. Moreover, Tropodiaptomus species show a noteworthy variability in some of the morphological characters considered of prime importance in diaptomid taxonomy, and the presence of cryptic or pseudocryptic species is likely. Thus, through a geographically-wide sampling in Thailand, we aimed to investigate the local diversity of the genus and to compare the morphological and molecular diversity pattern based on mitochondrial and nuclear genes sequences. DNA taxonomy was also implemented in order to check whether the Tropodiaptomus lineages were independent species according to the “evolutionary genetic species concept”. Six Tropodiaptomus morphospecies were found, three of which are putative species new to Science pending a formal description. The finding of such a high incidence of undescribed species stresses the existence of a significant “Linnean shortfall” affecting Thai diaptomids. The molecular results showed that most of the studied species could be identified consistently with their morphology-based taxonomy. However, Tropodiaptomus vicinus and T. cf. lanaonus showed a high level of genetic diversity, suggesting that traditional morphological techniques might be inadequate for correctly assessing their taxonomical status.

The complete chloroplast genome of Ficus pumila, a functional plant in East Asia

Ficus pumila L. is a climbing fig commonly used as an ornamental plant. In this study, we sequenced and assembled the complete chloroplast genome of F. pumila. The complete chloroplast genome of F. pumila is 160,248 bp in length which includes a pair of inverted repeats (IRs) of 25,871 bp separated by a large single-copy (LSC) region of 88,405 bp and a small single-copy (SSC) region of 20,101 bp. The overall guanine–cytosine (GC) content of F. pumila cp genome is 35.98%, while the corresponding values of LSC, SSC, and IR sequences are 33.65, 29.05, and 42.65%, respectively. The phylogenetic tree was shown to be consistent with the traditional morphology-based taxonomy of Moraceae. Five plants from the genus Ficus formed a well-supported monophyletic clade with 100% bootstrap value, and F. pumila is closely related to F. hirta, F. carica, and F. racemosa, with a support value of 97%. The complete chloroplast of F. pumila contributes to the growing number of chloroplast genomes for phylogenetic and evolutionary studies in Moraceae.

Fast and Reliable Identification of Ammonia Phylotypes T1, T2 and T6 Using a Stereomicroscope: Implication for Large-Scale Ecological Surveys and Monitoring Programs

Among benthic foraminifera, the genus Ammonia is characterized by high morphological variability which makes it particularly challenging to recognize using traditional morphology-based taxonomy. Despite the joint efforts made by both molecular and morphological taxonomists, it is still hard to identify different phylotypes based on their morphology. A new method was developed recently to discriminate three NE Atlantic phylotypes of Ammonia (T1, T2, and T6). This method is based on two morphometrical parameters using scanning electron microscope (SEM) images (i.e., the average pore diameter and the elevation of sutures on the spiral side), resulting individuals being correctly assigned to their phylotype in more than 90% of cases. In this study, we assess the possibility of implementing these criteria using a stereomicroscope. Phylotype assignations by SEM and stereomicroscopic identifications are in accordance for 62.6% of the scrutinized foraminifera and increase up to 79.5% when only the phylotype T6 is considered. Though the stereomicroscopic identification of Ammonia phylotypes based on these two morphological parameters needs to be cross-validated using molecular tools, this approach noticeably allows the identification of an individual 3 to 7 times faster than using a SEM. The ratio between accuracy and efficiency, an issue that is also attributable to the use of the rose Bengal staining method, suggests prioritizing the use of stereomicroscope identifications in large foraminiferal surveys. Finally, in the context that Ammonia phylotype T6 potentially being an alien species in Europe, this method will help to quickly identify Ammonia phylotypes; hence contributing to monitor the presence of T6 in different regions and then, offering interesting research perspectives to assess the timing and/or the progression of the possible invasion.