Cynthia del Carmen Penaflor Diaz and Dr. Keith A. Crandall, Integrative Biology
Introduction:
Freshwater crayfishes are taxonomically distributed among three families; two Northern Hemisphere families, Astacidae and Cambaridae and one Southern Hemisphere family, Parastacidae,which includes the Astacoides species. The phylogenetic relationships among the freshwater crayfish families and their relationships to lobster-like ancestors has generated considerable debate for at least 100 years. Because of this ongoing debate, the phylogenetic position of Parastacidae is unresolved. This study represents the first step in exploring the genetic resources of the endangered Malagasy Astacoides crayfish, and is the first large-scale phylogenetic analysis of this complex group. The results of this project will provide a phylogenetic estimate of relationships among the Madagascar Astacoides species. This phylogenetic framework will provide the necessary background for assessing future plans for preserving biodiversity of the threatened crayfishes from Madagascar.
Materials and Methods:
Crayfish Sampling:
Samples from the Astacoides species were collected from rivers throughout Madagascar. There is a very little information on the distribution and diversity of Astacoides species in Madagascar so this work may lead to the discovery of new species
Extracting and Sequencing:
DNA has been extracted from leg tissue using a cell lysis protocol. The Polymerase chain reaction (PCR) was utilized to amplify the 16S,12S and COI gene regions of mitochodrial DNA. The sequences have been edited and aligned using the computer program Sequencher 3.1.
Data Analysis:
Editing and aligning was done using MacClade. The protein-coding sequences (COI) was translated into amino acids to check the reading frame. The computer program PAUP* was used to estimate the evolutionary relationships between the different species using parsimony. Support of monophyletic groups was evaluation by bootstrapping.
Results and Conclusions:
Results:
Three mitochondrial genes, 12S, 16S, and COI, were sequences for 50 samples of Astacoides crawfish from throughout Madagascar. Parsimony analyses were
only performed on the COI data because further work on alignment of the ribosomal genes is needed. The COI phylogeny indicates that the Malagasy Astacoides
crawfish group into 3 well-supported clades with 90-100%bootstprap support.
Conclusions:
This result suggests that only three of the four species of Astacoides crawfish should be recognized. Specifically, our COI phylogeny argues for merging Ast. betsiloensis with
Ast. caldwelli. Phylogengetic analyses of the remaining two genes are needed to verify the COI result. Furthermore, population structure is quite distinct in each species. There
appears to be much gene flow in grannulus clade and some subdivision in the crosneri and betsiloensis clades. Ecologically this makes sense as the crosneri are burrowing
crayfish and therefore less prone to migration and the betsiloensis, while sharing habitat with grannulus, tend to be much larger and much fewer in number. Because they are larger, they will have a more fragmented habitat since large boulder areas are interspersed with riffle areas in the streams. Smaller population sizes naturally leads to increased subdivision.