- Email: [email protected]
Molecular evidence for the monophyly of Tenrecidae: a reply to Asher
MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 26 (2003) 331–332 www.elsevier.com/locate/ympev
Reply
Molecular evidence for the monophyly of Tenrecidae: a reply to Asher Asher argues that he ‘‘did not seriously question’’ the integrity of Tenrecidae and should not be cited for the Tenrecidae paraphyly hypothesis. In citing Asher (1999), we never implied that this was the only alternative, or even the preferred alternative that emerged from his analyses. We merely stated that ‘‘four of eight analyses presented by Asher (1999) challenge the monophyly of Tenrecidae and instead suggest that a subfamily of tenrecs, Tenrecinae, are more closely related to chrysochlorids (golden moles) than to other tenrecs, creating a paraphyletic Tenrecidae’’ (Douady et al., 2002, p. 358). In the Introduction section of his paper, Asher (1999, p. 233) puts the tenrecid monophyly hypothesis in the category of ‘‘entirely or partially untested.’’ In his Discussion section, and in light of his own results that show ‘‘occasional paraphyletic incursions’’ of the three golden mole genera, Asher (1999, p. 239) states that ‘‘The question now becomes does this relationship (e.g., between potamogalines and tenrecines) exclude the Chrysochloridae. Future analyses that include fossil and sequence data (for example) promise to help resolve this question.’’ We disagree that we misrepresented AsherÕs hypothesis of Malagasy tenrec paraphyly and explicitly stated (Douady et al., 2002, p. 360) that AsherÕs (1999) ‘‘conclusion was mainly supported by a close PotamogalinaeLimnogale (subfamily Oryzorictinae) relationship, but also by an association of the remaining Oryzorictinae with this clade to the exclusion of tenrecines.’’ AsherÕs (1999) analyses supported the former result in eight of eight analyses and the latter result in five of eight analyses. The statistical tests reported in Douady et al. (2002) are admittedly a test of only the weak version of the Malagasy tenrec paraphyly hypothesis espoused by Asher (1999), as they did not include Limnogale. Asher is correct that we did not explore issues such as ‘‘vagaries in sequence alignment,’’ exclusion of ‘‘alignment ambiguous regions,’’ and ‘‘treatment of gaps as information during phylogenetic reconstruction’’ and argues that we should ‘‘explore alternative homology assessments.’’ He further points to differences between our alignment and that of Stanhope et al. (1998) as evidence of the subjectivity of manually refined align-
ments. Several points are relevant here: (1) Our general strategy in previous papers has been to manually refine alignments and exclude alignment-ambiguous regions following the recommendation of Swofford et al. (1996). The rationale is that homology statements are less secure in regions where alignments are ambiguous. (2) It should come as no surprise that alignments and alignment-ambiguous regions differ between data sets [e.g., Stanhope et al. (1998) versus Douady et al. (2002)]. Manually refined alignments are admittedly subjective, but variation in taxon composition may result in alignment differences even when alignments are constructed with identical parameter settings and manual refinements are not employed. Similarly, the problem of alignment-ambiguous regions is expected to become more acute in studies that sample increasingly divergent taxa. (3) In the March issue of MPE in which Douady et al. (2002) appeared, manually refined alignments were the norm and only one out of 13 papers used nonmanual ‘‘objective’’ means of dealing with alignment ambiguities. New approaches for dealing with alignment ambiguous regions and gaps have value, but these methods also require subjective decisions, e.g., the range of alignment parameters to employ. Nevertheless, we reanalyzed our data after having produced new alignments using recently developed procedures for eliminating ambiguous sites (Table 1). We also performed an analysis on a modified version of our original data set that corrected the misalignment identified by Asher, which impacts only 12 of 3314 bp (0.36%) of the original data. Analyses based on these rRNA gene alignments confirm that the Douady et al. (2002) results are robust (Table 1). Tenrecid monophyly remained the best-supported hypothesis, even with the reduced number of nucleotides that were retained in the alignment generated by Proalign (Table 1). Homology statements are of fundamental importance in systematics. In molecular systematics, alignment construction and the identification and treatment of alignment-ambiguous regions are critical issues. However, homology problems and subjective decisions on how to recognize homology are not unique to molecular data. Asher (1999, p. 233) based his selection of
1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 5 5 - 7 9 0 3 ( 0 2 ) 0 0 3 3 7 - 8
332
Reply / Molecular Phylogenetics and Evolution 26 (2003) 331–332
Table 1 Ambiguity treatment and bootstrap support variation Published
ME ME MP ML ML
GTR LogDet TN TN + C + I
Manually corrected
SOAP
Monophyly
Paraphyly
Monophyly
Paraphyly
Monophyly
Paraphyly
Monophyly
ProAlign Paraphyly
94 95 80 85 93
5 3 8 10 6
94 94 70 78 91
4 4 13 13 5
92 92 80 84 92
6 5 5 6 2
78 81 70 64 69
14 12 11 14 18
Note. Published (2063 bp), manually corrected (2063 bp), SOAP (2027 bp), and ProAlign (1287 bp) correspond to four different alignments of the 12S–16S data used in Douady et al. (2002). ‘‘Published,’’ refers to the ‘‘original’’ alignment of Douady et al. (2002). ‘‘Manually corrected’’ is the same alignment, but incorporating a correction of the error highlighted in AsherÕs letter (Fig. 1). The ‘‘SOAP’’ alignment was generated using SOAP v.1.1 (L€ oytynoja and Milinkovitch, 2001) with 25 different settings and employing weighted matrix, gap penalties from 11 to 19, by steps of 2, extension penalties from 3 to 11, also by steps of 2, and 100% conservation (method derived form Gatesy et al., 1993). ‘‘ProAlign’’ alignment used Hidden Markov Models for a probabilistic multiple alignment. It was carried out with ProAlign v. 0.1 a 5 (L€ oytynoja and Milinkovitch, 2002). For computational reasons, the data were aligned as two independent halves using default parameters and a bandwidth of 201. All sites where alignment probability was above 90% were subsequently merged. Values in monophyletic and paraphyletic columns correspond to bootstrap values favoring tenrecid monophyly and tenrecid paraphyly, respectively, the latter with Tenrec and Echinops closer to Amblysomus than to Micropotamogale. ME GTR, minimum evolution with a general time reversible distance; ME LogDet, minimum evolution with a log determinant distance; MP, maximum parsimony; ML TN, maximum likelihood under a Tamura–Nei model; C þ I, allowance of rate heterogeneity across sites and a proportion of invariant sites. For detailed phylogenetic methods, see Douady et al. (2002).
morphological characters ‘‘primarily on three factors: (1) use in phylogenetic systematics by previous authors, (2) my own observations of relevant differences across taxa, and (3) ability to accurately categorize into discrete states.’’ We commend Asher for explicitly stating his criteria for character selection; such statements are often lacking in morphological studies. However, ‘‘relevant differences across taxa’’ and the ‘‘ability to accurately categorize characters into discrete states’’ are intrinsically subjective filters for excluding potential morphological character data and for determining which morphological features are incorporated into a character matrix.
Acknowledgment We thank Rob Asher for bringing the alignment error to our attention and for stimulating this discussion.
References Asher, R.J., 1999. A morphological basis for assessing the phylogeny of the ‘‘Tenrecoidea’’ (Mammalia, Lipotyphla). Cladistics 15, 231– 252. Douady, C.J., Catzeflis, F., Kao, D.J, Springer, M.S., Stanhope, M.J., 2002. Molecular evidence for the monophyly of Tenrecidae (Mammalia) and the timing of the colonization of Madagascar by Malagasy tenrecs. Mol. Phylogenet. Evol. 22, 357–363. Gatesy, J., DeSalle, R., Wheeler, W., 1993. Alignment-ambiguous nucleotide sites and the exclusion of systematic data. Mol. Phylogenet. Evol. 2, 152–157.
L€ oytynoja, A., Milinkovitch, M.C., 2001. SOAP, cleaning multiple alignments from unstable blocks. Bioinformatics 17, 573–574. L€ oytynoja, A., Milinkovitch, M.C., 2002. ProAlign, a probabilistic multiple alignment program. http://dbm.ulb.ac.be/ueg/ProAlign/. Stanhope, M.J., Waddell, V.G., Madsen, O., de Jong, W.W., Hedges, S.B., Cleven, G.C., Kao, D., Springer, M.S., 1998. Molecular evidence for multiple origins of the Insectivora and for a new order of endemic African mammals. Proc. Natl. Acad. Sci. USA 95, 9967–9972. Swofford, D.L., Olsen, G.P., Waddell, P.J., Hillis, D.M., 1996. Phylogenetic inference. In: Hillis, D.M., Moritz, C., Mable, B.K. (Eds.), Molecular Systematics. Sinauer, Sunderland, MA, pp. 407– 492.
Christophe J. Douady Department of Biochemistry and Molecular Biology Dalhousie University, Halifax Nova Scotia, Canada Francois Catzeflis Laboratoire de Paleontologie, CC 064, I.S.E.M. UMR 5554 CNRS, Place E. Bataillon Montpellier F-34095, France Mark S. Springer Department of Biology, University of California Riverside, CA 92521, USA E-mail address: [email protected] Michael J. Stanhope Bioinformatics, GlaxoSmithKline Pharmaceuticals 1250 South Collegeville Road, UP1345 Collegeville, PA 19426-0989, USA E-mail address: [email protected]
Reply
Molecular evidence for the monophyly of Tenrecidae: a reply to Asher Asher argues that he ‘‘did not seriously question’’ the integrity of Tenrecidae and should not be cited for the Tenrecidae paraphyly hypothesis. In citing Asher (1999), we never implied that this was the only alternative, or even the preferred alternative that emerged from his analyses. We merely stated that ‘‘four of eight analyses presented by Asher (1999) challenge the monophyly of Tenrecidae and instead suggest that a subfamily of tenrecs, Tenrecinae, are more closely related to chrysochlorids (golden moles) than to other tenrecs, creating a paraphyletic Tenrecidae’’ (Douady et al., 2002, p. 358). In the Introduction section of his paper, Asher (1999, p. 233) puts the tenrecid monophyly hypothesis in the category of ‘‘entirely or partially untested.’’ In his Discussion section, and in light of his own results that show ‘‘occasional paraphyletic incursions’’ of the three golden mole genera, Asher (1999, p. 239) states that ‘‘The question now becomes does this relationship (e.g., between potamogalines and tenrecines) exclude the Chrysochloridae. Future analyses that include fossil and sequence data (for example) promise to help resolve this question.’’ We disagree that we misrepresented AsherÕs hypothesis of Malagasy tenrec paraphyly and explicitly stated (Douady et al., 2002, p. 360) that AsherÕs (1999) ‘‘conclusion was mainly supported by a close PotamogalinaeLimnogale (subfamily Oryzorictinae) relationship, but also by an association of the remaining Oryzorictinae with this clade to the exclusion of tenrecines.’’ AsherÕs (1999) analyses supported the former result in eight of eight analyses and the latter result in five of eight analyses. The statistical tests reported in Douady et al. (2002) are admittedly a test of only the weak version of the Malagasy tenrec paraphyly hypothesis espoused by Asher (1999), as they did not include Limnogale. Asher is correct that we did not explore issues such as ‘‘vagaries in sequence alignment,’’ exclusion of ‘‘alignment ambiguous regions,’’ and ‘‘treatment of gaps as information during phylogenetic reconstruction’’ and argues that we should ‘‘explore alternative homology assessments.’’ He further points to differences between our alignment and that of Stanhope et al. (1998) as evidence of the subjectivity of manually refined align-
ments. Several points are relevant here: (1) Our general strategy in previous papers has been to manually refine alignments and exclude alignment-ambiguous regions following the recommendation of Swofford et al. (1996). The rationale is that homology statements are less secure in regions where alignments are ambiguous. (2) It should come as no surprise that alignments and alignment-ambiguous regions differ between data sets [e.g., Stanhope et al. (1998) versus Douady et al. (2002)]. Manually refined alignments are admittedly subjective, but variation in taxon composition may result in alignment differences even when alignments are constructed with identical parameter settings and manual refinements are not employed. Similarly, the problem of alignment-ambiguous regions is expected to become more acute in studies that sample increasingly divergent taxa. (3) In the March issue of MPE in which Douady et al. (2002) appeared, manually refined alignments were the norm and only one out of 13 papers used nonmanual ‘‘objective’’ means of dealing with alignment ambiguities. New approaches for dealing with alignment ambiguous regions and gaps have value, but these methods also require subjective decisions, e.g., the range of alignment parameters to employ. Nevertheless, we reanalyzed our data after having produced new alignments using recently developed procedures for eliminating ambiguous sites (Table 1). We also performed an analysis on a modified version of our original data set that corrected the misalignment identified by Asher, which impacts only 12 of 3314 bp (0.36%) of the original data. Analyses based on these rRNA gene alignments confirm that the Douady et al. (2002) results are robust (Table 1). Tenrecid monophyly remained the best-supported hypothesis, even with the reduced number of nucleotides that were retained in the alignment generated by Proalign (Table 1). Homology statements are of fundamental importance in systematics. In molecular systematics, alignment construction and the identification and treatment of alignment-ambiguous regions are critical issues. However, homology problems and subjective decisions on how to recognize homology are not unique to molecular data. Asher (1999, p. 233) based his selection of
1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 5 5 - 7 9 0 3 ( 0 2 ) 0 0 3 3 7 - 8
332
Reply / Molecular Phylogenetics and Evolution 26 (2003) 331–332
Table 1 Ambiguity treatment and bootstrap support variation Published
ME ME MP ML ML
GTR LogDet TN TN + C + I
Manually corrected
SOAP
Monophyly
Paraphyly
Monophyly
Paraphyly
Monophyly
Paraphyly
Monophyly
ProAlign Paraphyly
94 95 80 85 93
5 3 8 10 6
94 94 70 78 91
4 4 13 13 5
92 92 80 84 92
6 5 5 6 2
78 81 70 64 69
14 12 11 14 18
Note. Published (2063 bp), manually corrected (2063 bp), SOAP (2027 bp), and ProAlign (1287 bp) correspond to four different alignments of the 12S–16S data used in Douady et al. (2002). ‘‘Published,’’ refers to the ‘‘original’’ alignment of Douady et al. (2002). ‘‘Manually corrected’’ is the same alignment, but incorporating a correction of the error highlighted in AsherÕs letter (Fig. 1). The ‘‘SOAP’’ alignment was generated using SOAP v.1.1 (L€ oytynoja and Milinkovitch, 2001) with 25 different settings and employing weighted matrix, gap penalties from 11 to 19, by steps of 2, extension penalties from 3 to 11, also by steps of 2, and 100% conservation (method derived form Gatesy et al., 1993). ‘‘ProAlign’’ alignment used Hidden Markov Models for a probabilistic multiple alignment. It was carried out with ProAlign v. 0.1 a 5 (L€ oytynoja and Milinkovitch, 2002). For computational reasons, the data were aligned as two independent halves using default parameters and a bandwidth of 201. All sites where alignment probability was above 90% were subsequently merged. Values in monophyletic and paraphyletic columns correspond to bootstrap values favoring tenrecid monophyly and tenrecid paraphyly, respectively, the latter with Tenrec and Echinops closer to Amblysomus than to Micropotamogale. ME GTR, minimum evolution with a general time reversible distance; ME LogDet, minimum evolution with a log determinant distance; MP, maximum parsimony; ML TN, maximum likelihood under a Tamura–Nei model; C þ I, allowance of rate heterogeneity across sites and a proportion of invariant sites. For detailed phylogenetic methods, see Douady et al. (2002).
morphological characters ‘‘primarily on three factors: (1) use in phylogenetic systematics by previous authors, (2) my own observations of relevant differences across taxa, and (3) ability to accurately categorize into discrete states.’’ We commend Asher for explicitly stating his criteria for character selection; such statements are often lacking in morphological studies. However, ‘‘relevant differences across taxa’’ and the ‘‘ability to accurately categorize characters into discrete states’’ are intrinsically subjective filters for excluding potential morphological character data and for determining which morphological features are incorporated into a character matrix.
Acknowledgment We thank Rob Asher for bringing the alignment error to our attention and for stimulating this discussion.
References Asher, R.J., 1999. A morphological basis for assessing the phylogeny of the ‘‘Tenrecoidea’’ (Mammalia, Lipotyphla). Cladistics 15, 231– 252. Douady, C.J., Catzeflis, F., Kao, D.J, Springer, M.S., Stanhope, M.J., 2002. Molecular evidence for the monophyly of Tenrecidae (Mammalia) and the timing of the colonization of Madagascar by Malagasy tenrecs. Mol. Phylogenet. Evol. 22, 357–363. Gatesy, J., DeSalle, R., Wheeler, W., 1993. Alignment-ambiguous nucleotide sites and the exclusion of systematic data. Mol. Phylogenet. Evol. 2, 152–157.
L€ oytynoja, A., Milinkovitch, M.C., 2001. SOAP, cleaning multiple alignments from unstable blocks. Bioinformatics 17, 573–574. L€ oytynoja, A., Milinkovitch, M.C., 2002. ProAlign, a probabilistic multiple alignment program. http://dbm.ulb.ac.be/ueg/ProAlign/. Stanhope, M.J., Waddell, V.G., Madsen, O., de Jong, W.W., Hedges, S.B., Cleven, G.C., Kao, D., Springer, M.S., 1998. Molecular evidence for multiple origins of the Insectivora and for a new order of endemic African mammals. Proc. Natl. Acad. Sci. USA 95, 9967–9972. Swofford, D.L., Olsen, G.P., Waddell, P.J., Hillis, D.M., 1996. Phylogenetic inference. In: Hillis, D.M., Moritz, C., Mable, B.K. (Eds.), Molecular Systematics. Sinauer, Sunderland, MA, pp. 407– 492.
Christophe J. Douady Department of Biochemistry and Molecular Biology Dalhousie University, Halifax Nova Scotia, Canada Francois Catzeflis Laboratoire de Paleontologie, CC 064, I.S.E.M. UMR 5554 CNRS, Place E. Bataillon Montpellier F-34095, France Mark S. Springer Department of Biology, University of California Riverside, CA 92521, USA E-mail address: [email protected] Michael J. Stanhope Bioinformatics, GlaxoSmithKline Pharmaceuticals 1250 South Collegeville Road, UP1345 Collegeville, PA 19426-0989, USA E-mail address: [email protected]