Phylogenetic Analysis of Freshwater of Massachusetts: The ______

A Major Qualifying Project

Submitted to the Faculty

of

WORCESTER POLYTECHNIC INSTITUTE

in partial fulfillment of the requirements for the

Degree of Bachelor of Science

by

______Dilbar Ibrasheva Approved by:

______Professor Michael A. Buckholt Professor Lauren Mathews January 13, 2011 Contents Abstract ...... 3 Acknowledgements ...... 4 1 Introduction ...... 5 2 Background ...... 7 2.1 The Concept ...... 7 2.2 Phylogeography and Population Genetics ...... 8 3 Materials and Methods ...... 13 3.1 Samples and DNA Extraction ...... 17 3.1.1 Samples ...... 17 3.1.2 DNA Extraction ...... 18 3.2 PCR and mt DNA Sequencing ...... 19 4 Results ...... 21 5 Discussion ...... 25 6 Works Cited ...... 28 7 Appendices ...... 34

Abstract

Knowing the invasive nature of several species of freshwater crayfish of the genus

Procambarus, it was predicted that some of the watersheds within Massachusetts area could be

affected by this species. For this study 14 organisms of Procambarus genus, but undetermined

species were collected from 5 different sites within the state. After performing a set of

experiments, phylogenetic data was gathered. This study suggests that all of the organisms

collected either belong to one species – Procambarus acutus that is not considered to be an

invasive species, or are hybrids of both invasive Procambarus clarkii and Procambarus acutus.

Phylogenetic analysis grouped all of the organisms of undetermined species with only one

crayfish, which was a Procambarus acutus that originated from the Cape Fear River, Randolph

County, North Carolina. It was predicted that the specimen collected within Massachusetts could

have possibly been related to the specimen from North Carolina. However, further investigation

of larger size of population of both Procambarus clarkii and Procambarus acutus is necessary in order to fully understand the phylogenetic relationship between these species.

Acknowledgements

I would like to thank Professor Lauren Mathews and Professor Michael Buckholt for their guidance, time, patience and continuous support throughout the whole project.

I also want to thank Professor Destin Heilman, Professor Jill Rulfs, Abbie White and the whole Biology and Biotechnology Department at WPI for all the help, personal support and encouragement.

In addition, I would like to thank Ryan Clinton, Jack Sternal, Andy Nemeth and John

Burford for their help in the Project Lab.

1 Introduction

In the past years a significant amount of global research was conducted in the area of

phylogeography and conservational biology of freshwater crayfish (Bondar et al., 2005; Holdich,

2002; Jones et al., 2007). This study focuses on phylogeography of freshwater crayfish of the genus Procambarus that accounts for more than half of the total of 300 species of cambarid crayfish (Hobbs, 1981). 14 organisms of this genus were collected at 5 different sites in

Massachusetts. By identifying particular species of these organisms, and determining their

relationship to other Procambarus species, it became possible to obtain preliminary information

on the invasive nature of the freshwater crayfish of this genus. A study of spatial behavior of

Procambarus clarkii indicates that this species is invasive (Gherardi et al., 2000). Originating

from south-central part of the USA (Hobbs, 1972), it managed to escape from aquaculture

enclosures and succesffully establish in northern and central Italy with the breeding populations

(Gherardi et al., 2000). Introductions of new species to into native ecosystems can sometimes

lead to drastic changes in the local environment. According to Pimental, they can even cause loss

of species diversity and extinction of native species (Pimental et al., 2000).

It is worth mentioning that representatives of 3 out of 16 procambarid subgenera (Hobbs,

1974), are currently cultured and harvested (or simply harvested) on a commercial scale. These

are: Leconticambarus, Ortmannicus and Scapulicambarus. The major commercial specias are the

red swamp crayfish, Procambarus (Scapulicambarus) clarkii (Girard, 1852), the eastern white

river crayfish, Procambarus (Ortmannicus) acutus acutus (Girard, 1852), and the gulf white

river crayfish, Procambarus (Ortmannicus) zonangulus (Hobbs & Hobbs, 1990). Even though, the native distribution of Procambarus clarkii is north – eastern Mexico and

the south – central part of the USA, including Texas, Alabama, Tennessee and Illinois (Hobbs,

1972), it has been widely within and outside of the USA (Hobbs, 1972; Holdich, 2002).

Procambarus acutus species are naturally endemic to Texas, Louisiana, Missisippi and Alabama

(Huner, 1998). However, the white river crayfish has also been cultured eastward from Louisiana

to the Atlantic coast northwoard to Maine (Holdich, 2002). The fact that both of these species

have been cultured extensively throught the US, causes difficulties in understanding the origins

of the 14 organisms of the genus Procambarus that were used for this study.

Based on the phenotypical traits of these 14 crayfish, it was predicted that they are either

Procambarus clarkii or Procambarus acutus. However, to determine the exact species, it was

necessary to perform further genetic analysis. The result of this analysis was later used to gain

the insights into phylogenetic relationship between these organisms and to attempt to establish their original geographical location.

2 Background

2.1 The Species Concept

Most commonly the term ‘species’ is defined as a taxonomical rank which involves organisms that are able to interbreed and produce fertile progeny. However, even current scientists and philosophers have trouble agreeing upon the universal definition of this concept.

The roots of this so-called ‘species problem’, a debate between researches on identifying and classifying species and considering species, a level of biological organization, can be traced back to the 19th and 20th centuries (Hey, 2001). Even before Darwin’s “Origin of Species by Means of

Natural Selection or the Preservation of Favoured Races in the Struggle of Life” (Darwin, 1859)

various philosophers such as Aristotle and Theophrastus made attempts to give a full description

of species. They believed that the seeds of plants of one species could give rise to plants of other

species (Mayr, 1982). At that moment the concept of Typological species was at the forefront of

the debates (Yong & ZheKun, 2010).

Since that moment a lot of species models have been suggested in history. Various

scientists, such as Mayr (1982), Davis and Heywood (1963), Stuessy (1990), and Mayden

(1997), who managed to collect 22 species concepts, attempted to give an overview of the

definition of species. Nevertheless, based on the works of Wheeler and Meier (2000), it can be

concluded that the debate on the definition of species reached its climax at the end of the last

century. Four major species concepts were developed in this period of time: the Biological,

Hennigian, Phylogenetic and Evolutionary (Wheeler & Meier, 2000). However, several recent

studies conducted by biologists, such as Wu (Wu, 2001)and de Queiroz (de Queiroz, 2005)

indicate that nowadays even more and more new ways of defining the concept of species are

being developed. The species concept was influenced a lot by three major innovations in evolutionary

biology (Yong & ZheKun, 2010). The first one was shown by Darwin’s “Origin of Species by

Means of Natural Selection or the Preservation of Faboured Races in the Structure of Life”

(Darwin, 1859), as mentioned earlier. The major conclusion in his fundamental book was the

morphological species concept, which states that all currently living species originate from one

common ancestor through an evolutionary process known as natural selection (Darwin, 1859).

This interpretation of species differed drastically from the popular ones at that moment, which

were Essentialist and Creationist elucidations (Yong & ZheKun, 2010). Charles Darwin thought

that even though the term ‘species’ should not be referred to as a particular class of nature, it should be retained in biology and used by taxonomists (Ereshefsky, 2009). Later, a second innovation was introduced by Mayr and Dobzhansky, who expanded the already existing term

‘species’ by pointing out the need of taking into account reproductive isolation when studying speciation and species (Mallet, 2010). In 1942, Ernst Mayr introduced a set of new terms, the

Biological Species Concept (BSC) and the Phylogenetic Species Concept (PSC), which characterize populations as reproductive groups of individuals that inhabit a particular space in a certain period of time and that share a common gene pool (Coyne, 1994; Mayr, 1942 & 1982).

The third innovation was introduced by Hennig (Hennig, 1965), who brought mathematical methods into evolutionary biology that allowed biologists to define species with the aspect to phylogenetic systematics.

2.2 Phylogeography and Population Genetics

In order to understand better the consequences of invasion or introduction of exotic species

into the native ecosystem, it is necessary to understand several crucially important aspects of

phylogeography, population genetics and molecular ecology. The term phylogeography, which essentially means the phylogenetic analysis of data obtained

from organisms in relation to their current geographical distribution, was first introduced by

Avise in 1987 (Avise et al., 1987). Depending on a subject of study, Hickerson describes two

distinct types of phylogeography: single species phylogeographic studies that primarily focuses

on species determination and identification of hybrid zones, historical hybridization events,

geographic determinants of isolation and cases of introgression, and multi-species

phylogeographic studies that for the most part utilize tools of comparative biology to study the

influence of historical events on present patterns in biodiversity (Hickerson et al., 2009). These historical processes along with genetic responses are a major subject of study of population genetics.

The term ‘population’ can be defined as a group of organisms that belongs to the same species living in a particular area at the same time and with the ability to interbreed. Based on numerous studies of freshwater marine , including those concerning freshwater Galaxiid fishes and North American populations of Pink Salmon, Oncorhychus gorbuscha¸ (Waters &

Wallis, 2001; Aspinwall, 1974), it can be concluded that there are at least four major evolutionary processes that influence evolution and genetic composition in populations. These are gene flow, genetic drift, mutation, and natural selection (Halliburton, 2004).

Gene flow, an important concept frequently investigated in population genetics, population ecology and conservational biology, is also called gene migration and entails the transfer of alleles between populations. During the process of gene migration the allele frequencies between populations become homogenous. Due to the high gene flow, speciation between populations might be inhibited because of the absence of fixed alleles that could have been favorable for particular populations. This might inhibited the process of speciation. However, gene flow can also bring in novel alleles into a population or produce new

combinations of alleles and prevent random genetic drift. According to Freeland (2005), genetic

drift is defined as a process that gives rise to random variation in population’s allele frequences

from generation to generation. It can also be described as a random variation of allele

frequencies due to various spontaneous events such as random sampling of gametes. The

amount of genetic drift displayed is size dependent. In the long run genetic drift induces

decreased levels of heterozygosity and loss of alleles within the population, which results in

population divergance or separation. Genetic drift can be exemplified by a population bottleneck.

A population bottleneck implies a rapid reduction in population usually caused by natural

disasters, therefore reducing the overall level of genetic diversity, by creating narrowed sample

of allele frequencies (Halliburton, 2004). As described in a study involving invasive freshwater

snail Physa acuta (Bousset et al., 2004) various evolutionary events involve population

expansion, population bottlenecks, migration and variance. The effects of the size of the

bottleneck and the ability of the population to recover on the long-term genetic diversity of the population on species that have been introduced by humans to a new geographical location have been studied as well. These introductions usually result in a specific type of a population bottleneck known as a founder effect. The founder effect can be described by the fact that only

part of the genetic diversity of the source population will be carried on by the founders of the

new population (Freeland, 2005).

The relationships between populations of freshwater organisms have been known to be

formed by important geological changes in aquatic systems (Bernatchez & Wilson, 1998). This

dependence of genetic composition of populations on geological changes has been studied

extensively on populations of freshwater fishes in North America. One of the major events that significantly affected genetic diversity of aquatic organisms was the climate change during the

Pleistocene epoch of the Cenozoic Era (Near et al., 2001). The movement of several ice sheets towards south during that period left northern areas almost completely uninhabitable, thus forcing northern taxa to change their locations (Bernatchez & Wilson, 1998). It is also known that freshwater organisms followed multiple dispersal routes into the previously glaciated areas in the north following the global warming that happened at the end of Pleistocene (Mandrak &

Crossman, 1992). These significant climate changes are hypothesized to be responsible for facilitated dispersal of freshwater organisms in Europe that was triggered by the receding ice sheet later (Freeland et al., 2004). Additionally, it was reported that Pleistocene climate change gave rise to a rapid genetic change in upland organisms in other parts of North Amerca (Mayden,

1988; Near et al., 2001; Berendzen et al., 2003; Near & Keck, 2005: Ray et al., 2006). However, species native to the bottom land seemed to have the opposite effects from this event. Studies indicate that very little population sturcture has been determined across the Mississippi River watershed (Nedbal & Philipp, 1994; Avise, 2004).

Not only historical geographical events affect the relationship between and among populations. Intrinsic effects, involving habitat preference and the ability of the organisms to disperse among habitats, play an important role as well (Wares & Turner, 2003; Bohonak, 1999;

Bilton et al., 2001; Berg et al., 2007). Multiple studies indicate that dispersal capabilities is the major determinant of the among-population genetic structure for the freshwater organisms

(Bilton et al., 2001; Miller et al., 2002).

It can be concluded from previously mentioned studies that geographic barriers and distance have a direct affect on the gene flow within and among populations. Freshwater organisms like fish and many types of crayfish are generally restricted to their aquatic systems, therefore traveling between the watersheds does not occur. According to Fetzner (2003), even if two different populations of crayfish live in the geographical proximity, but in separate rivers, it does not mean that are close in linear river distance. This type of geographical isolation usually leads to population subdivision, causing new subpopulations to emerge. Different factors including the lack of gene flow and mutations lead to genetic divergence (Halliburton, 2004).

Additionally, it is important to take into account behavioral and life history features of the populations. In the case of population genetics studies of Cherax destructor, an Australian freshwater crayfish, these traits played a major role in differentiation of populations of two distinct watersheds: northern and southern (Hughes & Hillyer , 2003; Nguyen et al., 2005).

Phylogeographical analysis of subpopulations of shovel-nosed salamanders in southern

Appalachians performed by Jones (2006) showed that the degree of differentiation between subpopulations was much higher across the Eastern Continental Divide that separated two rivers into single basins than the degree of differentiation among the subpopulations within the the river basins.

Populations of two different types of freshwater crayfish (subterranean and local surface dwelling) from southeaster America were studied and compared (Buhay & Crandall , 2005).

Findings indicate that despite the fact that the cave crayfish is more isolated than the surface species, the subterranean crayfish had higher levels of both gene flow and genetic diversity. It was also determined that the surface dwelling species were showing a decline in genetic variablity.

3 Materials and Methods

By definition, molecular ecology is a branch of evolutionary biology that deals with finding ways to answer major questions of ecology by applying methods of molecular phylogenetics, molecular population genetics and genomics, and other molecular analyses. In

order to approach those questions and test hypotheses, molecular tools such as molecular genetic

markers are frequently used. Compared to other kinds of ecological measurements, molecular

markers and marker variations can give quantifiable and precise genetic data, which can be helpful for statistical comparisons. Even though application of molecular genetic markers for phylogenetic purposes requires a lot of training of the practioners and significant financial support, Avise (2004) recommended that molecular markers may be used for analyzing

controversial questions and problems of natural history and evolution.

Molecular markers are considered to be either polymorphic proteins or stretches of DNA

sequence that can be employed as indicators of genome – wide variation. Usually, these are

sections of the genome, of a very small relative to the whole genome of an organism, that are

chosen to represent bigger stretches of DNA. Each of these sections is referred to as a ‘locus’

that does not necessarily have to be a functional gene. However, not all sections of the genome

can be considered to be useful molecular markers. One of the key factors that gives a certain type

of molecular marker a higher preference value is its level of polymorphism which can vary from

zero to hundreds of alternative alleles over a single species’ range. Highly polymorphic markers

are very useful for behavioral studies, whereas markers with a moderate level of polymorphism

are widely used in population genetic analysis (Avise, 2004). However, for phylogeographic investigations like this study, mitochondrial genes are often employed, because of the advantages discussed later. Mitochondrial ribosomal genes such as 12S and 16S, as well as protein-coding genes such as cytochrome oxidase I (COI), have been extensively utilized in population genetic and systematic studies. Mitochondrial markers have been chosen over nuclear DNA markers in studies due to several reasons. According to Toon et al. (2009), mitochondrial markers are comparatively easy to isolate since the copy number of mitochondria present in tissues is relatively high. They are also inherited predominantly maternally in most species, which enables scientists to track maternal lineages back in time.

Various studies on mitochondrial DNA of Xenopus, humans, mice and mammals indicate maternal inheritance (Avise et al., 1987; Hutchison et al., 1974; Dawid & Blackler, 1972; Giles et al., 1980; Gyllensten et al., 1985). Even though the maternal mode of inheretance is considered to be the major one, enough research was completed to show that that way of passing on mitochondrial genes is not the only one. Studies have shown that this type of inheritance can take place in different species of mice and in Drosophila (Avise, 1991; Gyllensten et al., 1991;

Kondo et al., 1990). The most notable hereditary mode was determined to be ‘doubly uniparental’, meaning that females pass on their mitochondrial genes to both sons and daughters, whereas males transmit their mtDNA only to sons (Hoeh, 1991, 1997, 2002) (Zouros, 1992,

1994). All of these studies were focused on the inheritance mode of mitochondrial genes within a certain mollusk system. It has also been reported that in this system, genetic recombination between mtDNA molucules took place (Ladoukakis & Zouros, 2001). Although, thus far, the case of Mytilus mussels is the only known exception (Zouros et al., 2000). Nevertheless, the mtDNA is mostly maternally inhereted. During this process mtDNA molecules provide markers that are transmitted asexually through generations. Therefore, mtDNA genotypes are

called molecular clones or haplotypes.

Besides the fact that mitochondrial markers are mostly inhereted maternally, they are also haploid and thus are only a quarter of the effective population size of the nuclear genes (Avise,

2004). The fact that mitochondrial DNA is not highly conserved, it has a fast mutation rate,

which is very useful for studying phylogeny of different organisms.

Additionally, it takes noticeably less time for the initial set up of the experiments, and the

presence and availability of universal primers make mitochondrial markers favorable for studies

(Toon et al. , 2009). Moreover, a set of nucleotide sequences for these three mitochondrial genes

is already present in GenBank database, since they have been used extensivly for a large period

of time for molecular phylogenetic analysis, thus making further analyses easier.

In order to obtain information from DNA sequencing, it is essential to clone and amplify

the gene of interest from the biological source first. Due to its very specific nature, Polymerase

Chain Reaction (PCR) enables researches to amplify assayable amounts of desirable genes from

large and complex genomes (Freeland, 2005).

This technique was first described by Mullis and Faloona in 1987 (Mullis & Faloona,

1987). The PCR technique involves three major steps: denaturation of double-stranded DNA by

heating, annealing of the oligonucleotide primers to the regions flanking the gene of interest, and

primer extension, during which the complementary to the desirable gene strands are synthesized

with the aid of thermo stable Taq DNA Polymerase (Avise, 2004). These three consecutive steps

must be repeated at least twenty times. During each cycle the amount of target DNA almost

doubles in its quantity. Figure 1: Polymerase Chain Reaction from (Griffiths, 2000)

Figure 1 gives a basic overview of this procedure.

In this study, 16S rRNA mitochondrial gene from each specimen was amplified using Polymerase Chain Reaction, the PCR products were cleaned up and sent for sequencing to DNA Sequencing Facility at Cornell University.

3.1 Samples and DNA Extraction

3.1.1 Samples

All of the organisms were collected at five different sites within Massachusetts State between 2006 and 2008. The specimen were previously used for various studies, however the particular species of the specimen were never determined. All the information known about the species was collected and summarized in the table below (Table 1).

Table 1: Samples and Collection Sites

Sample Collection Site Date summer A M 109 2008 Birch Hill Dam, ACE, collection site 2, voucher 1 (Royalston, not B MA) specified Birch Hill Dam, ACE, collection site 2, voucher 2 (Royalston, not C MA) specified D M 7, Birch Hill Dam (Royalston, MA), voucher 8/19/2006 E M 103 7/9/2008 F M 103 7/9/2008 G M 103 7/9/2008 H M 103 7/9/2008 I M 98 6/20/2008 J M 98 6/20/2008 K M 98 6/20/2008 L M 98 6/20/2008 M M 98 6/20/2008 N M 98 6/20/2008

3.1.2 DNA Extraction

For this project 5-10 mg of muscle tissue was extracted from each individual’s claw or a

leg using Gentra’s Puregene Protocol: DNA Purification from Tissue Using the Gentra Puregene

Tissue Kit (Qiagen, 2010). The tissue was thoroughly ground placed in 300 µL of Cell Lysis

Solution and heated up at 65°C for 1 hour. Later, 1.5 µL of Puregene Proteinase K was added to

each of the tubes, and the tubes were inverted at least 25 times. Afterwards, the samples were

incubated at 55°C overnight for maximum yield. The next day 3 µL of RNase A Solution were

added to the tubes, the tubes were mixed by inverting 25 times and left to incubate at 37°C for 60

minutes. As soon as the sampels were taken out of the heat block, they were placed on ice for 1

minute. 100 µL of Protein Precipitation Solution were added to each of the tubes, and the samples were vortexed vigorously for 20 seconds at high speed. In order to separate proteins

from the samples, the tubes were centrifuged for 3 minutes at 13,000 x g. The precipitated

proteins did not form a tight pellet in some samples, thus, those tubes were incubated on ice for 5

minutes and were centrifuged at the same parameters again. The supernatant was later added to

14 fresh tubes, each containing 300 µL of isopropanol. These tubes were inverted gently at least

50 times, and soon after centrifuged at 13,000 x g for 1 minute. The supernatant was carefully

discarded. After the tubes were drained by inverting on KimWipes, they were left to air dry for 5

minutes. The samples were hydrated by adding 50 µL of TE Buffer and were vortexed at

medium speed for 5 seconds. In order to dissolve the DNA, the samples were incubated for 60

minutes at 65°C. Subsequently, these 14 tubes were tightly closed and left to incubate at room

temperature overnight with gentle shaking. In the morning, samples were were cooled down and

frozen at -20°C. Later, they would be thawed at 37°C before each use. To determine DNA concentration for each individual, it was decided to run out 1 µL of

DNA from each sample tube along with the hyperladder and 3 λ DNA samples with known concentrations: 10 ng/ µL, 100 ng/ µL and 200 ng/ µL on 1.5 % agarose gel. 1 µL of DNA from each sample tube and 3 λ DNA samples were mixed with Syber Green, glycerol and loading buffer, and the gel was run at 100 Volts for about 2 hours.

The concentrations of sample DNA were predicted to be about 100 ng/ µL.

3.2 PCR and mt DNA Sequencing

Most of the protocols suggest that 20 ng of DNA is used, however, considering that it was difficult to determine the right concentrations for all the 14 samples, it was decided to use approximately 100ng instead. To amplify approximately 550 base pair fragment of 16S rRNA mitochondrial gene, two primers were used:

16S – 1472: 5’-AGATAGAAACCAACCTGG-3’

16S – L2: 5’-TGCCTGTTTATCAAAAACAT-3’

PCR reactions were carried out in 20 µL tubes. Each tube contained about 100 ng of sample DNA, each primer at 1.2 µM, dNTPs at 2 mM each, 10X buffer at a final concentration of 1X, MgCl2 at 2 µM, 13.8 µL of autoclaved deionized water, and 0.4 µL of Taq polymerase enzyme.

The negative control tubes had no genomic DNA, 1 µL of autoclaved deionized water was used instead.

Reaction conditions were as follows:

1. 95°C for 2 minutes 2. 95°C for 30 seconds 3. 48°C for 30 seconds run for 40 cycles 4. 72°C for 60 seconds 5. Repeat steps 2-4 6. 72°C for 2 minutes 7. 10°C forever

Subsequently, 2% agarose gel electrophoresis was used to ensure the amplification of 16S gene in each of the reactions. The samples (5 µL of each, mixed with 0.5 µL of loading buffer) were run along with a 100 base pair hyperladder that was used a molecular weight standard. The gel was run at 120 Volts for about 90 minutes, and was later post-stained with Ethidium

Bromide. Gel electrophoresis revealed that out of 14 initial samples, only 12 contained successfully amplified 16S rRNA mitochondrial gene.

Before the sequencing step of the experiment, it was necessary to perform the clean up of

PCR products. This was done by following the Exonuclease I – Shrimp Alkaline Phosphatase clean up of PCR products protocol (Nucleics, 2010). To create the 1X ExoSAP Mix I used 0.025

µL of Exonuclease I, 0.250 µL of SAP and 9.724 µL Milli Q Water per sample. 10 µL of the 1X

ExoSAP mix were added to each sample tube containing the PCR products. These samples were later incubated at 37°C for 30 minutes and then at 95°C for 5 minutes in the PCR thermocycler.

Afterwards, the samples were taken out of the machine and stored at -20°C.

To obtain the sequencing data, the cleaned up PCR samples were sent to DNA Sequencing

Facility, Biotechnology Resource Center at Cornell University. In order to more accurate and precise results, the PCR samples for each individual organism were split in two, 10 µL in each tube. 1 µL of 16S – 1742 primer was added to the first tube, whereas 1 µL of 16S – L2 primer was added to the second. 24 tubes for 12 organisms were packaged and sent to Cornell.

4 Results

The phylogenetic analysis of 12 organisms of the genus Procambarus was performed based on the sequencing data of 16S rRNA mitochondrial gene. Due to the fact that PCR products for each individual were sequenced both in forward and reverse directions, both of the sequences had to be compared and fixed. Using multiple software programs, including SeqAssem,

Geneious and BioEdit the obtained sequences were assembled and edited, and a single contig for each organism was created.

To identify the species of these 12 organisms and to obtain more information on their site of origin and common ancestors, a set of known 16S rRNA mitochondrial gene sequences from

GenBank were used.

All the published sequences for 16S rRNA gene for Procambarus clarkia and Procambarus acutus and single sequences of all other species of the genus Procambarus were used for data analysis.

The table below shows the particular Procambarus clarkii and Procambarus acutus sequences used.

Table 2: Published Sequences: Procambarus clarkii and Procambarus Acutus

GenBank Assesion Number Collection site FJ619803.1 - AF235990.1 - EF012350.1 (33.2; –87.55) DQ666844.1 - EF012351.1 (32.1586; –104.2889) AF436040.1 (29.697; –95.8977) (north of Procambarus EF012352.1 Fulshear) clarkii GQ168838.1 Supermarket Germany : Saxony FJ619805.1 - FJ619804.1 - EF012353.1 Downs Prairie (35.3416 ;–94.043) Procambarus EF012354.1 Fourche Creek, (34.656 ; –92.422) acutus EU433915.1 Randolph Cape Fear River, NC

Unfortunately, the collection site data not for all of the Procambarus clarkii and

Procambarus acutus specimen was published online or was available.

Table 3: Published Sequences Procambarus spp.

Specimen GenBank Accession Number Procambarus alleni FJ619802.1 Procambarus curdi EF012344.1| Procambarus digueti AY214435.1 Procambarus fallax FJ619801.1 Procambarus gibbus EU433916.1 Procambarus liberorum EF012333.1 Procambarus nigrocinctus EF012345.1 Procambarus ouachitae EF012356.1 Procambarus pecki EU433911.1 Procambarus reimeri EF012343.1 Procambarus tenuis EF012349.1 Procambarus toltecae AY214438.1

The list of sequences of different species of the Procambarus genus and their GenBank accession numbers are shown above in the Table 3.

However, Clustal W alignment of all the 12 organisms and all of the organisms specified in the table above was made using BioEdit software. Afterwards, a phylogenetic tree of these sequences was built. For this purpose, MEGA software was utilized.

Figure 2: Phylogenetic Tree

The full size image of this phylogenetic tree is located in Appendices.

To verify the common origin of all of the 12 organisms and their correlation to a

particular Procambarus acutus organism (GeneBank Assesion number: EU433915.1) and to identify the species, Sequence Identity matrix was constructed, using MEGA software.

Table 4: Sequence Identity Matrix

The 96% - 97% between the sequences is represented by light blue color, 97%-98% by

light green and 99% or above match by orange color. As can be clearly seen from the table

above, the relation between the Procambarus acutus organism (GeneBank Assesion number:

EU433915.1) has the highest % match.

The full size image of the sequence identity matrix is located in the Appendices of this

report.

It was also determined that none of the sequences of these 12 organisms are absolutely

identical, which leads to a conclusion that each of them is of a different haplotype. 5 Discussion

One of the goals of this study was to determine the species of freshwater crayfish collected at five different sites in Massachusetts. A priori it was determined that the organisms belong to

Procambarus genus, although, particular species could not be identified simply based on morphological traits. It was suggested that they were either Procambarus clarkii or Procambarus acutus.

According to the sequencing data involving sequence identity matrix (Table 5) and phylogenetic analysis (Figure 2), it can be suggested that all the organisms either belong to

Procambarus acutus species, or are hybrids of both Procambarus acutus and Procambarus clarkii. The sequence identity matrix (Table 5) indicates that all of the 12 organisms have the highest % match with the Procambarus acutus specimen from Cape Fear River, Randolph

County, North Carolina. Phylogenetic analysis also grouped these individuals together.

Since the sequence editing was completed by eye, it was decided to build another phylogenetic tree, using a different software program and unedited sequences of the organisms of study. The software I used was Geneious. The original names of the contigs were modified by the default settings of the software. The table below represents the original sample name and its corresponding reference number for the unedited sequence.

Table 5: Unedited Sequences original specimen A B D E F G H I J K L M unedited sequence reference # (contig #) - 2 3 4 5 6 7 8 9 10 11 12

The result of phylogenetic analysis is shown in Figure 3. Figure 3: Phylogenetic Tree of unedited sequences

Even though the sequences were not trimmed and were not aligned, Geneious software still grouped all of the 12 crayfish of undetermined species together with the Procambarus acutus sample from North Carolina, proving that no significant errors and mistakes during the initial data analysis, that could affect the grouping of the specimen. Even though the data for this study indicates that all of the specimen are related to the

Procambarus acutus crayfish from North Carolina, further investigation is required. Increasing

the pool of organisms of different populations of Procambarus genus from various watersheds

within Massachusetts would help to build more accurate phylogenetic trees. It is also important

to take into account that the crayfish of Procambarus genus could be easily introduced by

people, due to the fact that these organisms are used for commercial purposes. As a result, a lot

of new places in the US could be experiencing the founder effect, where invasive species like

Procambarus clarkii start a completely new population.

This study gave a preliminary insight on phylogenetics of freshwater crayfish of

Procambarus genus in Massachusetts, and hopefully, will be useful for further research of organisms of this genus.

6 Works Cited

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7 Appendices

Contig - B Contig - J Contig - F Contig - G Contig - M Contig - H Contig - E Contig - I - Copy Contig - D Contig - L - Copy Contig - K Contig - A gi|187711179|gb|EU433915.1| Procambarus acutus 16S ribosomal RNA gene partial sequence mitochondrial gi|237824184|gb|FJ619805.1| Procambarus acutus isolate KC987 16S ribosomal RNA gene partial sequence mitochondrial gi|237824183|gb|FJ619804.1| Procambarus acutus isolate KC986 16S ribosomal RNA gene partial sequence mitochondrial gi|28974307|gb|AY214438.1| Procambarus toltecae 16S ribosomal RNA gene partial sequence mitochondrial gene for mitochondrial product gi|121591984|gb|EF012354.1| Procambarus acutus haplotype 2 16S ribosomal RNA gene partial sequence gi|237824180|gb|FJ619801.1| Procambarus fallax isolate KC3839 16S ribosomal RNA gene partial sequence mitochondrial gi|121591986|gb|EF012356.1| Procambarus ouachitae haplotype 2 16S ribosomal RNA gene partial sequence gi|237824181|gb|FJ619802.1| Procambarus alleni isolate KC3852 16S ribosomal RNA gene partial sequence mitochondrial gi|121591982|gb|EF012352.1| Procambarus clarkii haplotype 2 16S ribosomal RNA gene partial sequence gi|121591980|gb|EF012350.1| Procambarus clarkii haplotype 1 16S ribosomal RNA gene partial sequence gi|110348229|gb|DQ666844.1| Procambarus clarkii 16S ribosomal RNA gene partial sequence mitochondrial gi|121591981|gb|EF012351.1| Procambarus clarkii haplotype 3 16S ribosomal RNA gene partial sequence gi|237824182|gb|FJ619803.1| Procambarus clarkii isolate KC1156 16S ribosomal RNA gene partial sequence mitochondrial gi|7621469|gb|AF235990.1| Procambarus clarkii KC837 16S ribosomal RNA gene partial sequence mitochondrial gene for mitochondrial product gi|187711180|gb|EU433916.1| Procambarus gibbus 16S ribosomal RNA gene partial sequence mitochondrial gi|121591979|gb|EF012349.1| Procambarus tenuis haplotype 1 16S ribosomal RNA gene partial sequence gi|28974301|gb|AY214435.1| Procambarus digueti 16S ribosomal RNA gene partial sequence mitochondrial gene for mitochondrial product gi|187711175|gb|EU433911.1| Procambarus pecki 16S ribosomal RNA gene partial sequence mitochondrial gi|121591974|gb|EF012344.1| Procambarus curdi 16S ribosomal RNA gene partial sequence gi|121591975|gb|EF012345.1| Procambarus nigrocinctus 16S ribosomal RNA gene partial sequence gi|121591963|gb|EF012333.1| Procambarus liberorum haplotype 22 16S ribosomal RNA gene partial sequence gi|121591973|gb|EF012343.1| Procambarus reimeri haplotype 1 16S ribosomal RNA gene partial sequence

0.01

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