A synthetic phylogeny of freshwater crayfish: insights for conservation

Freshwater ecosystems, despite covering only 0.8% of the Earth's surface, support nearly 6% of all described species. These ecosystems face numerous threats, including overexploitation, pollution, habitat destruction, and invasive species, leading to alarming extinction rates (around 4% per decade). Crayfish are a vital component of these endangered ecosystems, playing a central ecological role and often holding economic and cultural significance for human communities. They are considered keystone species, impacting stream communities through consumption, predation, sediment bioturbation, and organic matter processing. However, over 30% of the world's crayfish species are endangered, highlighting the urgency for conservation efforts. Crayfish represent an ideal group for phylogenetic synthesis due to their robust taxonomy and extensive previous phylogenetic research. This study integrates existing phylogenetic studies with a curated taxonomy to create a comprehensive synthetic tree for all freshwater crayfish. This tree is then used to map IUCN endangered species status, estimate divergence and extinction rates, and perform EDGE and HEDGE analyses to identify high-priority conservation targets. The study showcases the power of combining taxonomy and synthetic phylogeny to inform conservation assessments based on phylogenetic diversity and endangerment.

The introduction cites numerous studies emphasizing the biodiversity crisis in freshwater ecosystems and the importance of crayfish within those systems. These studies highlight the ecological roles of crayfish, their endangerment status, and previous phylogenetic work on specific crayfish groups. The authors establish the need for a comprehensive phylogenetic framework to guide conservation efforts by referencing earlier, smaller-scale phylogenetic studies of crayfish and conservation prioritization efforts that utilize phylogenetic information in other taxa. The existing literature provided the basis for the current study, which builds upon these previous efforts by creating a much broader, more inclusive phylogenetic analysis.

The study employed a phylogenetic synthesis approach, combining multiple sources of phylogenetic information with an established taxonomy. This differs from supertree methods by explicitly incorporating the underlying taxonomic framework, allowing for the visualization and tracing of conflicts between different sources. A curated taxonomy from the Open Tree of Life project (OTT2.2) was used, incorporating data from various sources and resolving taxonomic inconsistencies. Twenty published phylogenetic studies, representing various taxonomic levels and molecular data, were uploaded to The Open Tree of Life Study Curator. These studies were merged using a tree alignment graph approach in treemachine to create the synthetic tree. To obtain branch lengths and divergence time estimates, a separate crayfish phylogram was constructed using PHLAWD, incorporating GenBank sequence data from across the group. The analysis included portions of three mitochondrial genes (12S, 16S, COI) and two nuclear genes (18S, 28S). Sequences were aligned using MAFFT, and poorly aligned regions were removed using GBLOCKS. Two lobster species served as outgroups. A maximum-likelihood (ML) phylogeny was estimated in GARLI, and branch support was assessed using bootstrapping. A chronogram was then estimated using penalized likelihood in R8S, incorporating six fossil calibrations. To assess diversification rates, the researchers used a Monte Carlo Constant Rates Test and birth-death chronogram simulations to account for missing taxa, and employed ML in TREEPAR to estimate time-dependent speciation and extinction rates. Finally, the study performed EDGE and HEDGE analyses, integrating IUCN Red List status with phylogenetic diversity (PD) metrics (Faith’s PD and PSV) calculated in MESQUITE and picante to rank species based on evolutionary distinctiveness and endangerment. Phylogenetic species variability (PSV) was calculated to examine broad geographical patterns of diversity and endangerment within the context of pre-established freshwater ecozones (FADA ecozones).

The synthetic tree included 719 terminal taxa, comprising all 590 described species of freshwater crayfish and additional representatives from population-level data. The molecular phylogeny, based on a 5259 bp alignment, strongly supported the sister clade relationship between Northern and Southern Hemisphere taxa but showed paraphyly for Cambaridae and Astacidae, with only Parastacidae being strongly supported as monophyletic. The analysis of diversification rates revealed that diversification was not constant through time, with a three-rate birth-death model providing the best fit, indicating shifts in diversification rates at 20 Ma and 4 Ma. The death parameter increased towards the present, while the birth parameter initially decreased and then increased recently. The highest turnover rate occurred in the most recent period. PD and PSV calculations across FADA ecozones showed variation in diversity patterns, with the Nearctic region exhibiting the highest PD and the Australasian region displaying the highest PSV. The Afrotropical region showed the lowest diversity. EDGE and HEDGE scores were calculated for species with IUCN values. *Fallicambarus hortoni* had the highest EDGE and HEDGE scores. The top 10 species with the highest EDGE and HEDGE scores included critically endangered and endangered species predominantly from Australia and a few North American cave species.

The findings confirm the need for a comprehensive reassessment of crayfish taxonomy, particularly in North America, where rapid radiation has resulted in paraphyletic genera and families. In contrast, the longer terminal branches observed in the Southern Hemisphere, potentially due to historical biogeographic events, result in more monophyletic genera. The study's phylogeny, although representing 60% of known species, is incomplete, potentially overestimating EDGE and HEDGE scores. Traditional phylogenetic markers are insufficient to resolve many relationships; the authors advocate for the use of genomic data (e.g., ultraconserved elements, transcriptomes) to improve phylogenetic resolution. Diversification rate analyses indicate non-constant rates through time, correlating with major geological events. Future work should integrate ecological, morphological, geographic, and phylogenetic data to investigate diversification and extinction rates more precisely.

This study presents a significant advance in understanding freshwater crayfish phylogeny and informing conservation efforts. The synthesis of taxonomic and phylogenetic information provides a powerful framework for identifying high-priority conservation targets. The authors emphasize the need for future work to refine the taxonomy, improve phylogenetic resolution using genomic data, and incorporate additional ecological and environmental data to understand diversification patterns more comprehensively. The incomplete sampling of species in this study is a significant limitation that highlights the ongoing need for taxonomic and phylogenetic research.

The study acknowledges the limitations of its findings. Incomplete taxonomic sampling (around 60% of described species) affects the accuracy of the EDGE and HEDGE scores and the interpretation of diversification analyses. The use of traditional molecular markers produced insufficient resolution for some clades, calling for more extensive genomic sequencing in future research. The chronogram estimation relies on point estimates, with inherent uncertainty associated with these estimates. Also, the authors note that the current taxonomy needs major revisions, as evidenced by the paraphyletic nature of several genera and the lack of monophyly in the families, except for Parastacidae.