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Lorenzo Angelus Fernandez Participation credit – species delimitation On Canvas, please download the file “Cyerce_COI_aligned.fas”, our original fasta file containing COI sequences from all Caribbean specimens of Cyerce . When I collected these specimens, they were all one nominal (named, or recognized) species called “ Cyerce antillensis ”. When I performed species delimitation, the results indicated this was actually a cryptic species complex, meaning a group of related but distinct species that no one had told apart yet. Using this file, follow along with the lecture and see how we can use two methods of species delimitation to group individuals into distinct bins, or clusters, that presumably correspond to what we would recognize as nameable “species”. 1] ABGD – automated barcode gap detection Go to the website: https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html Copy-paste the .fasta file contents (COI sequences) into the box. Choose a model of DNA substitution to correct your genetic distances. Change any parameters if you want (pmin, pmax, etc). Hit return. Question 1: How many species did it find? 7 species Partition 1 : found 7 groups (prior maximal distance P= 0.001000) Partition 2 : found 7 groups (prior maximal distance P= 0.001995) Partition 3 : found 7 groups (prior maximal distance P= 0.003979) Partition 4 : found 7 groups (prior maximal distance P= 0.007937) Partition 5 : found 7 groups (prior maximal distance P= 0.015832) Partition 6 : found 7 groups (prior maximal distance P= 0.031581) Partition 7 : found 7 groups (prior maximal distance P= 0.062996) Click on “ here ” to see your results. 1 1

Question 2: Based on the histogram, what would you estimate is the barcoding gap – that is, the gap between intra-specific and inter-specific genetic distances? Does that agree with the gap calculated by ABGD? The barcoding gap is roughly ~0.058 or 5.8%, which does seem to agree with the calculations of ABGD and 7 groups/species. Click on one of the yellow dots to see the (a) barcoding gap, (b) grouping of individuals into species or “Groups”. Question 3: Based on the groups, are all named specimens grouping together? Are any specimens in groups that don’t match their names? Yes, the named species are grouping together. Tampa and Tarpon are grouped together in one group. Group 1 has over 5 species and outlier individuals Cy_antillensis_07SSal01 Cy_antillensis_10Bim01 Cy_antillensis_KM879888 Cy_antillensis_TXID1582513 Cy_antillensis_TXID1460625 in it. 2 2

Question 4: Now go back and change the pmin and pmax values to something else, or change the model of DNA substitution, and run a new analysis. Tell me the new parameters you used, and whether you got different results? New parameters: Partition 1 : found 7 groups (prior maximal distance P= 0.001000) Partition 2 : found 7 groups (prior maximal distance P= 0.001911) Partition 3 : found 7 groups (prior maximal distance P= 0.003652) Partition 4 : found 7 groups (prior maximal distance P= 0.006980) Partition 5 : found 7 groups (prior maximal distance P= 0.013338) Partition 6 : found 7 groups (prior maximal distance P= 0.025490) Partition 7 : found 7 groups (prior maximal distance P= 0.048714) The P value changes to varying extents but the number of groups remains the same, and the results on the histogram also appear to be the same. 2] ASAP – Assemble Species by Automatic Partitioning Go to the website: https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html Upload the .fasta file contents (COI sequences) by clicking the “Choose a file” button. Choose a model of DNA substitution to correct your genetic distances. Hit return. Question 5: How many species did ASAP find in the top-scoring partition? What was the P-value associated with this result? 29 9.60e-01 Click on “ list ” (at the right-most column) to download a text file with your results, including the individuals sorted into groups (=species). Partition 10 3 3

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Asap-Score: 12.000000 Proba: 9.580838e-01 Nb subsets with recursion:29 (without recursion: 28) ------------------------------------------------------------ Subset[ 1 ] n: 1 ;id: Cy_antillensis_Pan_MM51 Subset[ 2 ] n: 18 ;id: Cy_antillensis_Pan_MM56B Cy_antillensis_Lee_U4 Cy_antillensis_07Swe12 Cy_antillensis_Pan_MM56C Cy_antillensis_06Dom05 Cy_antillensis_10Swe02 Cy_antillensis_07Swe18 Cy_antillensis_10Swe04 Cy_antillensis_07Swe15 Cy_antillensis_07SSal01 Cy_antillensis_06Dom01 Cy_antillensis_06Dom03 Cy_antillensis_10Swe03 Cy_antillensis_07Swe11 Cy_antillensis_07Swe14 Cy_antillensis_Pan_MM57 Cy_antillensis_Pan_MM56E Cy_antillensis_TXID1460625 Subset[ 3 ] n: 1 ;id: Cy_antillensis_TXID1582513 Subset[ 4 ] n: 1 ;id: Cy_antillensis_Lee_U5 Subset[ 5 ] n: 1 ;id: Cy_antillensis_06Dom04 Subset[ 6 ] n: 1 ;id: Cy_antillensis_10Swe01 Subset[ 7 ] n: 1 ;id: Cy_antillensis_07Swe17 Subset[ 8 ] n: 1 ;id: Cy_antillensis_10Bim01 Subset[ 9 ] n: 1 ;id: Cy_antillensis_KM879888 Subset[ 10 ] n: 1 ;id: Cy_cristallina_FL_B91 Subset[ 11 ] n: 4 ;id: Cy_cristallina_FL_B94 Cy_cristallina_FL_MM97 Cy_cristallina_FL_B99 Cy_cristallina_FL_MM98 Subset[ 12 ] n: 2 ;id: Cy_cristallina_FL_B108 Cy_cristallina_FL_B109 Subset[ 13 ] n: 1 ;id: Cy_cristallina_FL_MM99 Subset[ 14 ] n: 1 ;id: Cy_nicholasi_04Pan01 Subset[ 15 ] n: 2 ;id: Cy_nicholasi_07Dom01 Cy_nicholasi_07LSS01 Subset[ 16 ] n: 8 ;id: Cy_nicholasi_10Bim02 Cy_nicholasi_07Stir03 Cy_nicholasi_07Stir05 Cy_nicholasi_07Stir09 Cy_nicholasi_07Swe10 Cy_nicholasi_07Swe02 Cy_nicholasi_07Swe09 Cy_nicholasi_07Swe06 Subset[ 17 ] n: 1 ;id: Cy_nicholasi_10Bim03 Subset[ 18 ] n: 1 ;id: Cy_nicholasi_07LSS04 Subset[ 19 ] n: 1 ;id: Cy_nicholasi_07Stir04 Subset[ 20 ] n: 1 ;id: Cy_nicholasi_07Stir06 Subset[ 21 ] n: 1 ;id: Cy_nicholasi_07Stir10 Subset[ 22 ] n: 1 ;id: Cy_nicholasi_07Swe04 Subset[ 23 ] n: 1 ;id: Cy_nicholasi_07Swe07 Subset[ 24 ] n: 10 ;id: Cy_piercei_Tampa_KM55 Cy_piercei_Tampa_KM56 Cy_piercei_TarponSp_KM66 Cy_piercei_TarponSp_KM68 Cy_piercei_Tampa_KM57 Cy_piercei_TarponSp_KM65 Cy_piercei_TarponSp_KM69 Cy_piercei_06Tam01 Cy_piercei_07Swe03 Cy_piercei_07Pla01 Subset[ 25 ] n: 1 ;id: Cy_piercei_07Stir02 Subset[ 26 ] n: 6 ;id: Cy_ellingsonorum_06Jam01 Cy_ellingsonorum_06Jam07 Cy_ellingsonorum_06Jam08 Cy_ellingsonorum_06Jam05 Cy_ellingsonorum_06Jam06 Cy_ellingsonorum_06Jam04 Subset[ 27 ] n: 1 ;id: Cy_ellingsonorum_06Jam03 Subset[ 28 ] n: 1 ;id: Cy_willetteorum_07Stir01 Subset[ 29 ] n: 8 ;id: Cy_browneveorum_09Gei01 Cy_browneveorum_12Sum04 Cy_browneveorum_12Sum01 Cy_browneveorum_S216_KM879893 Cy_browneveorum_12Sum03 Cy_browneveorum_10Gei01 Cy_browneveorum_12Sum02 Cy_browneveorum_S219_KM879892 Question 6: Did ASAP group individuals the same way ABGD did? 4 4

It grouped the individuals in a similar graph. As far as I can tell the values are the same between them Question 7: Scroll down the list to the partition with 8 species, and download that list. This tells you what ASAP thinks would be the most likely 8 th species, if there was another one in this collection of individuals. What specimen or group of specimens would be the 8 th species? Species 8: Partition 8 Asap-Score: 9.500000 5 5

Proba: 9.041916e-01 Nb subsets with recursion:32 (without recursion: 30) ------------------------------------------------------------ Subset[ 1 ] n: 1 ;id: Cy_antillensis_Pan_MM51 Subset[ 2 ] n: 18 ;id: Cy_antillensis_Pan_MM56B Cy_antillensis_Lee_U4 Cy_antillensis_07Swe12 Cy_antillensis_Pan_MM56C Cy_antillensis_06Dom05 Cy_antillensis_10Swe02 Cy_antillensis_07Swe18 Cy_antillensis_10Swe04 Cy_antillensis_07Swe15 Cy_antillensis_07SSal01 Cy_antillensis_06Dom01 Cy_antillensis_06Dom03 Cy_antillensis_10Swe03 Cy_antillensis_07Swe11 Cy_antillensis_07Swe14 Cy_antillensis_Pan_MM57 Cy_antillensis_Pan_MM56E Cy_antillensis_TXID1460625 Subset[ 3 ] n: 1 ;id: Cy_antillensis_Lee_U5 Subset[ 4 ] n: 1 ;id: Cy_antillensis_06Dom04 Subset[ 5 ] n: 1 ;id: Cy_antillensis_10Swe01 Subset[ 6 ] n: 1 ;id: Cy_antillensis_07Swe17 Subset[ 7 ] n: 1 ;id: Cy_antillensis_10Bim01 Subset[ 8 ] n: 1 ;id: Cy_antillensis_KM879888 Subset[ 9 ] n: 1 ;id: Cy_antillensis_TXID1582513 Subset[ 10 ] n: 1 ;id: Cy_cristallina_FL_B91 Subset[ 11 ] n: 4 ;id: Cy_cristallina_FL_B94 Cy_cristallina_FL_MM97 Cy_cristallina_FL_B99 Cy_cristallina_FL_MM98 Subset[ 12 ] n: 2 ;id: Cy_cristallina_FL_B108 Cy_cristallina_FL_B109 Subset[ 13 ] n: 1 ;id: Cy_cristallina_FL_MM99 Subset[ 14 ] n: 1 ;id: Cy_nicholasi_04Pan01 Subset[ 15 ] n: 2 ;id: Cy_nicholasi_07Dom01 Cy_nicholasi_07LSS01 Subset[ 16 ] n: 8 ;id: Cy_nicholasi_10Bim02 Cy_nicholasi_07Stir03 Cy_nicholasi_07Stir05 Cy_nicholasi_07Stir09 Cy_nicholasi_07Swe10 Cy_nicholasi_07Swe02 Cy_nicholasi_07Swe09 Cy_nicholasi_07Swe06 Subset[ 17 ] n: 1 ;id: Cy_nicholasi_10Bim03 Subset[ 18 ] n: 1 ;id: Cy_nicholasi_07LSS04 Subset[ 19 ] n: 1 ;id: Cy_nicholasi_07Stir04 Subset[ 20 ] n: 1 ;id: Cy_nicholasi_07Stir06 Subset[ 21 ] n: 1 ;id: Cy_nicholasi_07Stir10 Subset[ 22 ] n: 1 ;id: Cy_nicholasi_07Swe04 Subset[ 23 ] n: 1 ;id: Cy_nicholasi_07Swe07 Subset[ 24 ] n: 5 ;id: Cy_piercei_Tampa_KM55 Cy_piercei_Tampa_KM56 Cy_piercei_TarponSp_KM66 Cy_piercei_TarponSp_KM68 Cy_piercei_Tampa_KM57 Subset[ 25 ] n: 1 ;id: Cy_piercei_TarponSp_KM65 Subset[ 26 ] n: 1 ;id: Cy_piercei_TarponSp_KM69 Subset[ 27 ] n: 3 ;id: Cy_piercei_06Tam01 Cy_piercei_07Swe03 Cy_piercei_07Pla01 Subset[ 28 ] n: 1 ;id: Cy_piercei_07Stir02 Subset[ 29 ] n: 6 ;id: Cy_ellingsonorum_06Jam01 Cy_ellingsonorum_06Jam07 Cy_ellingsonorum_06Jam08 Cy_ellingsonorum_06Jam05 Cy_ellingsonorum_06Jam06 Cy_ellingsonorum_06Jam04 Subset[ 30 ] n: 1 ;id: Cy_ellingsonorum_06Jam03 Subset[ 31 ] n: 1 ;id: Cy_willetteorum_07Stir01 Subset[ 32 ] n: 8 ;id: Cy_browneveorum_09Gei01 Cy_browneveorum_12Sum04 Cy_browneveorum_12Sum01 Cy_browneveorum_S216_KM879893 Cy_browneveorum_12Sum03 Cy_browneveorum_10Gei01 Cy_browneveorum_12Sum02 Cy_browneveorum_S219_KM879892 6 6

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