We present Procrustean Approach to Cophylogeny (PACo), a novel statistical tool to test for congruence between phylogenetic trees, or between phylogenetic distance matrices of associated taxa. Unlike previous tests, PACo evaluates the dependence of one phylogeny upon the other. This makes it especially appropriate to test the classical coevolutionary model that assumes that parasites that spend part of their life in or on their hosts track the phylogeny of their hosts. The new method does not require fully resolved phylogenies and allows for multiple host-parasite associations. PACo produces a Procrustes superimposition plot enabling a graphical assessment of the fit of the parasite phylogeny onto the host phylogeny and a goodness-of-fit statistic, whose significance is established by randomization of the host-parasite association data. The contribution of each individual host-parasite association to the global fit is measured by means of jackknife estimation of their respective squared residuals and confidence intervals associated to each host-parasite link. We carried out different simulations to evaluate the performance of PACo in terms of Type I and Type II errors with respect to two similar published tests. In most instances, PACo performed at least as well as the other tests and showed higher overall statistical power. In addition, the jackknife estimation of squared residuals enabled more elaborate validations about the nature of individual links than the ParaFitLink1 test of the program ParaFit. In order to demonstrate how it can be used in real biological situations, we applied PACo to two published studies using a script written in the public-domain statistical software R.

Summary1. The concordance of evolutionary histories and extant species interactions provides a useful metric for addressing questions of how the structure of ecological communities is influenced by macro-evolutionary processes. 2. We introduce paco (v0.3.1), an R package to perform Procrustean Approach to Cophylogeny. This method assesses the phylogenetic congruence, or evolutionary dependence, of two groups of interacting species using both ecological interaction networks and their phylogenetic history. 3. We demonstrate the functionality of paco through its application to empirical host-parasite and plant-pollinator communities. 4. Although the package is intended to assess the phylogenetic congruence between groups of interacting species, the method is also directly applicable to other scenarios that may show phylogenetic congruence including historical biogeography, molecular systematics, and cultural evolution.

The inference of evolutionary relationships in the species-rich family Orchidaceae has hitherto relied heavily on plastid DNA sequences and limited taxon sampling. Previous studies have provided a robust plastid phylogenetic framework, which was used to classify orchids and investigate the drivers of orchid diversification. However, the extent to which phylogenetic inference based on the plastid genome is congruent with the nuclear genome has been only poorly assessed. METHODS:We inferred higher-level phylogenetic relationships of orchids based on likelihood and ASTRAL analyses of 294 low-copy nuclear genes sequenced using the Angiosperms353 universal probe set for 75 species (representing 69 genera, 16 tribes, 24 subtribes) and a concatenated analysis of 78 plastid genes for 264 species (117 genera, 18 tribes, 28 subtribes). We compared phylogenetic informativeness and support for the nuclear and plastid phylogenetic hypotheses.RESULTS: Phylogenetic inference using nuclear data sets provides well-supported orchid relationships that are highly congruent between analyses. Comparisons of nuclear gene trees and a plastid supermatrix tree showed that the trees are mostly congruent, but revealed instances of strongly supported phylogenetic incongruence in both shallow and deep time. The phylogenetic informativeness of individual Angiosperms353 genes is in general better than that of most plastid genes. CONCLUSIONS:Our study provides the first robust nuclear phylogenomic framework for Orchidaceae and an assessment of intragenomic nuclear discordance, plastid-nuclear tree incongruence, and phylogenetic informativeness across the family. Our results also demonstrate what has long been known but rarely thoroughly documented: nuclear and plastid phylogenetic trees can contain strongly supported discordances, and this incongruence must be reconciled prior to interpretation in evolutionary studies, such as taxonomy, biogeography, and character evolution.

Phylogenetic relationships inferred from multilocus organellar and nuclear DNA data are often difficult to resolve because of evolutionary conflicts among gene trees. However, conflicting or "outlier" associations (i.e., linked pairs of "operational terminal units" in two phylogenies) among these data sets often provide valuable information on evolutionary processes such as chloroplast capture following hybridization, incomplete lineage sorting, and horizontal gene transfer. Statistical tools that to date have been used in cophylogenetic studies only also have the potential to test for the degree of topological congruence between organellar and nuclear data sets and reliably detect outlier associations. Two distance-based methods, namely ParaFit and Procrustean Approach to Cophylogeny (PACo), were used in conjunction to detect those outliers contributing to conflicting phylogenies independently derived from chloroplast and nuclear sequence data. We explored their efficiency of retrieving outlier associations, and the impact of input data (unit branch length and additive trees) between data sets, by using several simulation approaches. To test their performance using real data sets, we additionally inferred the phylogenetic relationships within Neotropical Catasetinae (Epidendroideae, Orchidaceae), which is a suitable group to investigate phylogenetic incongruence because of hybridization processes between some of its constituent species. A comparison between trees derived from chloroplast and nuclear sequence data reflected strong, well-supported incongruence within Catasetum, Cycnoches, and Mormodes. As a result, outliers among chloroplast and nuclear data sets, and in experimental simulations, were successfully detected by PACo when using patristic distance matrices obtained from phylograms, but not from unit branch length trees. The performance of ParaFit was overall inferior compared to PACo, using either phylograms or unit branch lengths as input data. Because workflows for applying cophylogenetic analyses are not standardized yet, we provide a pipeline for executing PACo and ParaFit as well as displaying outlier associations in plots and trees by using the software R. The pipeline renders a method to identify outliers with high reliability and to assess the combinability of the independently derived data sets by means of statistical analyses.

Cophylogeny provides an appropriate setting to untie how the ecological and evolutionary facets of species interactions operate.

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Ligophorus mugilinus was originally described on the flathead mullet Mugil cephalus from the Gulf of Mexico. The species was later recorded in the Mediterranean Sea on the flathead mullet and in the Caribbean Sea on the white mullet Mugil curema. After examination of the type material and additional specimens of Ligophorus from the northwest Atlantic coast, the Caribbean Sea, and the Mediterranean and Black Sea, we observed morphological differences concerning haptoral and male copulatory structures between L. mugilinus from the northwest Atlantic and the forms from the Mediterranean and Black Seas. Accordingly, the latter specimens are described as a new species, Ligophorus mediterraneus n. sp. A redescription of L. mugilinus, including new morphometric data, is also given. The morphometric analysis of the Caribbean specimens suggested that their ascription to L. mugilinus is uncertain, but additional material is needed to clarify their specific identity. The results of this study, together with previous zoogeographical evidence, indicate that flathead mullets from different seas harbor different species complexes of Ligophorus. Given the coastal habits of the host, it is suggested that open oceans could act as geographical barriers favoring speciation within disjointed flathead mullet populations.

A comparative morphological study of specimens of Ligophorus spp. from Mugil cephalus in western Mediterranean and the Black Sea localities has been carried out, indicating the presence of 2 distinct forms, i.e., Ligophorus chabaudi and Ligophorus cephali n. sp. A detailed description of the latter and an up-to-date redescription of L. chabaudi are provided. The existence of these 2 morphological species was additionally supported by principal component analysis based on 19 metric characters of 87 specimens arranged in samples defined by parasite species and geographical locality. The analysis indicated consistent differences between species but not between localities; this pattern was well supported by jackknife procedures. Linear discriminant analyses showed that the main metric differences between the 2 species were attributable to the lengths of the dorsal and ventral bars and to the uncinulus length; use of the first 2 variables was enough to allocate all specimens studied to either form. The erection of L. cephali n. sp. raises the number of sympatric Mediterranean species of Ligophorus on M. cephalus to 3. The known geographical ranges of these species are compared.