Phylogenetic analysis of the Monocotylidae (Monogenea) inferred from 28S rDNA sequences

The current classification of the Monocotylidae (Monogenea) is based on a phylogeny generated from morphological characters. The present study tests the morphological phylogenetic hypothesis using molecular methods. Sequences from domains C2 and D1 and the partial domains C1 and D2 from the 28S rDNA gene for 26 species of monocotylids from six of the seven subfamilies were used. Trees were generated using maximum parsimony, neighbour joining and maximum likelihood algorithms. The maximum parsimony tree, with branches showing less than 70% bootstrap support collapsed, had a topology identical to that obtained using the maximum likelihood analysis. The neighbour joining tree, with branches showing less than 70% support collapsed. differed only in its placement of Heterocotyle capricornensis as the sister group to the Decacotylinae clade. The molecular tree largely supports the subfamilies established using morphological characters. Differences are primarily how the subfamilies are related to each other. The monophyly of the Calicotylinae and Merizocotylinae and their sister group relationship is supported by high bootstrap values in all three methods, but relationships within the Merizocotylinae are unclear. Merizocotyle is paraphyletic and our data suggest that Mycteronastes and Thaumatocotyle, which were synonymized with Merizocotyle after the morphological cladistic analysis, should perhaps be resurrected as valid genera. The monophyly of the Monocotylinae and Decacotylinae is also supported by high bootstrap values. The Decacotylinae, which was considered previously to be the sister group to the Calicotylinae plus Merizocotylinae, is grouped in an unresolved polychotomy with the Monocotylinae and members of the Heterocotylinae. According to our molecular data, the Heterocotylinae is paraphyletic. Molecular data support a sister group relationship between Troglocephalus rhinobatidis and Neoheterocotyle rhinobatidis to the exclusion of the other species of Neoheterocotyle and recognition of Troglocephalus renders Neoheterocotyle,le paraphyletic. We propose Troglocephalus incertae sedis. An updated classification and full species list of the Monocotylidae is provided. (C) 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Morphometric and cladistic analyses of the phylogeny of Macropodinium (Ciliophora : Litostomatea : Macropodiniidae)

Phylogenetic studies of the genus Macropodinium were conducted using two methods; phenetics and cladistics. The phenetic study of morphometrics suggested that the genus could be divided into 3 groups attributable mostly to cell size and shape. The cladistic study also split the genus into 3 groups related to cell size but groups were further distinguished by patterns of ornamentation. Reconciliation of both approaches revealed considerable congruence, however, it also suggested the existence of convergences in the phenetic study and a lack of resolution in the cladistic study. The morphological diversity of Macropodinium is probably due to evolutionary trends such as increasing body size, allometry and polymerisation of structures. None of these trends, however, was uniformly directional and differential effects were observed in different regions of the phylogenetic tree. Comparison of the phylogeny of Macropodinium to a consensus phylogeny of the macropodids revealed limited incongruence between the 2 trees. The ciliate groups could be related to 2 host groups; the wallaby genera and the kangaroo and wallaroo subgenera. The association with these host groups may be the result of phyletic codescent, ecological resource tracking or a combination of both. Further studies of both host and ciliate phylogeny are necessary to resolve these effects.

The status of the genera Hysterolecithoides Yamaguti, 1934, Neotheletrum Gibson & Bray, 1979 and Machidatrema Leon-Regagnon, 1998 (Digenea : Hemiuroidea), including a description of M. leonae n. sp from Australian waters

A diagnosis is given for the lecithasterid genus Hysterolecithoides Yamaguti, 1934, which is now found to have two to six (possibly seven) vitelline masses. The species H. frontilatus (Manter, 1969) is returned to the genus, having been considered a member of the bunocotylid genus Neotheletrum by recent authors. It is redescribed from Siganus nebulosus, Moreton Bay, and S. doliatus, Lizard Island, Great Barrier Reef and New Caledonia, with emphasis on the presence of Juel's organ, a uterine seminal receptacle and the blind sac associated with the genital atrium. It differs from its congeners in the trajectory of the pars prostatica which recurves dorsally to the sinus-sac. Oligolecithoides Shen, 1982 is synonymised with Hysterolecithoides and O. trilobatus Shen, 1982 is synomised with H. epinepheli Yamaguti, 1934. Machidatrema Leon-Regagnon, 1998 is diagnosed, and found to be close to Hysterolecithoides, but differs in the lack of a blind-sac projecting from the dorsal genital atrium, by its tandem testes, the coiling of the uterus between the testes and the ovary, and the ventral excretory pore. M. leonae n. sp. is described from Siganus fuscescens, S. lineatus, S. doliatus, S. corallinus, S. vulpinus and Scarus globiceps at Heron Island, Queensland. It differs from its closest congener, M. akeh, in the muscular and tegumental flap over the genital pore and details of the terminal genitalia. M. chilostoma (Machida, 1980) and M. kyphosi (Yamaguti, 1970) are redescribed from Kyphosus vaigiensis from Heron Island. Neotheletrum Gibson & Bray, 1979 is diagnosed: it differs from Hysterolecithoides in its confluent excretory arms, blind seminal receptacle (no Juel's organ) and uniformly tripartite vitellarium. A cladistic analysis suggests that M. chilostoma and M. kyphosi are not best accommodated in Machidatrema, that Machidatrema (sensu stricto) is monophyletic and that Hysterolecithoides is paraphyletic. Hysterolecithoides and Machidatrema are considered hysterolecithine lecithasterids, whilst Neotheletrum is retained as an opisthadenine bunocotylid.

Tormopsolus attenuatus n. sp (Digenea : Acanthocolpidae) from Seriola hippos (Perciformes : Carangidae), Western Australia, with some observations on the relationships in the genus

A new acanthocolpid, Tormopsolus attenuatus n. sp., is described from the carangid Seriola hippos from waters off the coast of SW Australia. A cladistic analysis of the genus using 31 morphological and metrical characters indicates that the closest species is T. asiatica Parukhin, 1976 status amend. (previously T. orientalis asiatica). T. attenuatus differs from T. asiatica in its smaller overall dimensions, more attenuated outline, relatively shorter post-testicular region, vitelline interruption at the level of the ovary, papillae on the oral sucker and, possibly, much longer genital atrium. Other similar species, T. orientalis Yamaguti, 1934 and T. medius Reimer, 1983, differ in having interruptions of the vitelline fields at the level of both testes and at the level of the ovary.

A review of the family Enenteridae Yamaguti, 1958 (Digenea), with descriptions of species from Australian waters, including Koseiria huxleyi n. sp.

The family Enenteridae is reviewed, with keys to the genera and species and diagnoses of the family and genera, based on a cladistic analysis utilising 44 characters. Subfamilies are not recognised. Descriptions of the following taxa from Australian marine teleosts are given: Enenterum mannarense from Kyphosus sydneyanus, SW Australia, E. elongatum from Kyphosus sydneyanus, SW Australia (these two species are distinguished by the number of oral lobes and the ovary to anterior testis distance), Koseiria huxleyi n. sp. from Chaetodontoplus meredithi, Great Barrier Reef (this new species is distinguished by the vitellarium reaching into the forebody, the infundibuliform terminal oral sucker, the unlobed ovary and the distinct post-oral ring), Koseiria xishaense from Kyphosus cinerascens and K. vaigiensis, Great Barrier Reef, Cadenatella isuzumi from Kyphosus cinerascens and K. vaigiensis, Great Barrier Reef, and C. pacifica (Yamaguti, 1970) n. comb. [was Jeancadenatia] from Kyphosus cinerascens and K. vaigiensis, Great Barrier Reef. The genus Jeancadenatia is considered a synonym of Cadenatella, and the new combination C. dollfusi (Hafeezullah, 1980) is formed. Members of the family are parasitic mainly in herbivorous fishes with a few genera and species from non-herbivorous fishes.

Phylogenetic revision of Agapophytinae subf.n. (Diptera : Therevidae) based on molecular and morphological evidence

Agapophytinae subf.n. is a highly diverse lineage of Australasian Therevidae, comprising eight described and two new genera: Agapophytus Guerin-Meneville, Acupalpa Krober, Acraspisa Krober, Belonalys Krober, Bonjeania Irwin & Lyneborg, Parapsilocephala Krober, Acatopygia Krober, Laxotela Winterton & Irwin, Pipinnipons gen.n. and Patanothrix gen.n. A genus-level cladistic analysis of the subfamily was undertaken using sixty-eight adult morphological characters and c. 1000 base pairs of the elongation factor-1 alpha (EF-1 alpha) protein coding gene. The morphological data partition produced three most parsimonious cladograms, whereas the molecular data partition gave a single most parsimonious cladogram, which did not match any of the cladograms found in the morphological analysis. The level of congruence between the data partitions was determined using the partition homogeneity test (HTF) and Wilcoxon signed ranks rest. Despite being significantly incongruent in at least one of the incongruence tests, the partitions were combined in a simultaneous analysis. The combined data yielded a single cladogram that was better supported than that of the individual partitions analysed separately. The relative contributions of the data partitions to support for individual nodes on the combined cladogram were investigated using Partitioned Bremer Support. The level of support for many nodes on the combined cladogram was non-additive and often greater than the sum of support for the respective nodes on individual partitions. This synergistic interaction between incongruent data partitions indicates a common phylogenetic signal in both partitions. It also suggests that criteria for partition combination based solely on incongruence may be misleading. The phylogenetic relationships of the genera are discussed using the combined data. A key to genera of Agapophytinae is presented, with genera diagnosed and figured. Two new genera are described: Patanothrix with a new species (Pat. skevingtoni) and Pat. wilsoni (Mann) transferred from Parapsilocephala, and Pipinnipons with a new species (Pip. kroeberi). Pipinnipons fascipennis (Krober) is transferred from Squamopygin Krober and Pip. imitans (Mann) is transferred from Agapophytus. Agapophytus bicolor (Krober) is transferred from Parapsilocephala. Agapophytus varipennis Mann is synonymised with Aga, queenslandi Krober and Aga. flavicornis Mann is synonymised with Aga. pallidicornis (Krober).

Phylogenetic revision of Agapophytus Guerin (Diptera : Therevidae : Agapophytinae)

Morphologically diverse and species-rich, the endemic Australasian genus Agapophytus is revised. Eleven previously described species are redescribed and twenty-nine species are described for the first time: A. adonis, sp. nov., A. annamariae, sp. nov., A. antheliogynaion, sp. nov., A. asprolepidotos, sp. nov., A. atrilaticlavius, sp. nov., A. biluteus, sp. nov., A. borealis, sp. nov., A. caliginosus, sp. nov., A. cerrusus, sp. nov., A. chaetohypopion, sp. nov., A. chrysosisyrus, sp. nov., A. decorus, sp. nov., A. dieides, sp. nov., A. discolor, sp. nov., A. eli, sp. nov., A. fenestratum, sp. nov., A. galbicaudus, sp. nov., A. labifenestellus, sp. nov., A. laparoceles, sp. nov., A. lissohoplon, sp. nov., A. lyneborgi, sp. nov., A. notozophos, sp. nov., A. novaeguineae, sp. nov., A. pallidicrus, sp. nov., A. palmulus, sp. nov., A. paramonovi, sp. nov., A. septentrionalis, sp. nov., A. yeatesi, sp. nov. and A. zebra, sp. nov. All 40 species of Agapophytus were compared in a cladistic analysis with three species of Acupalpa Krober using 134 states across 58 adult morphological characters. The analysis resulted in 36 most parsimonious trees with a length of 240 steps. The phylogenetic relationships of the species of Agapophytus are discussed with three main clades recognised: A. dioctriaeformis clade, A. australasiae clade and A. queenslandi clade.

Phylogenetic revision of Australian members of the Allomethus genus group (Diptera : Pipunculidae)

The Australian species of Allomethus and Claraeola are revised and include one described species, Claraeola erinys (Perkins), and five new species: Allomethus unicicolis sp. n., Claraeola cyclohirta sp. n., C. sicilis sp. n., C. spargosis sp. n., and C. yingka sp. n.. Claraeola hylaea (Perkins) is proposed to be a synonym of C. erinys (Perkins). A key to species is provided and male and female genitalia are illustrated. The Australian species are placed phylogenetically into a world context using available taxa within the Allomethus genus group. The phylogenetic relationships are discussed in light of a cladistic analysis involving 22 taxa and 60 characters.

Molecular evidence for the common origin of snap-traps among carnivorous plants

The snap-trap leaves of the aquatic waterwheel plant (Aldrovanda) resemble those of Venus' flytrap (Dionaea), its distribution and habit are reminiscent of bladderworts (Utricularia), but it shares many reproductive characters with sundews (Drosera). Moreover, Aldrovanda has never been included in molecular phylogenetic studies, so it has been unclear whether snap-traps evolved only once or more than once among angiosperms. Using sequences from nuclear 18S and plastid rbcL, atpB, and matK genes, we show that Aldrovanda is sister to Dionaea, and this pair is sister to Drosera. Our results indicate that snap-traps are derived from flypaper-traps and have a common ancestry among flowering plants, despite the fact that this mechanism is used by both a terrestrial species and an aquatic one. Genetic and fossil evidence for the close relationship between these unique and threatened organisms indicate that carnivory evolved from a common ancestor within this caryophyllid clade at least 65 million years ago.

Molecular phylogenetics of Lentibulariaceae inferred from plastid rps16 Intron and trnL-F DNA sequences: implications for character evolution and biogeography

Phylogenetic relationships among 75 species of Lentibulariaceae, representing the three recognized genera, were assessed by cladistic analysis of DNA sequences from the plastid rps16 intron and the trnL-F region. Sequence data from the two loci were analyzed both separately and in combination. Consensus trees from all analyses are congruent, and parsimony jackknife results demonstrate strong support for relationships both between and within each of the three demonstrably monophyletic genera. The genus Pinguicula is sister to a Genlisea-Utricularia clade, the phylogenetic structure within this clade closely follows Taylor's recent sectional delimitations based on morphology. Three principal clades are shown within Utricularia, with the basal sections Polypoinpholyx and Pleiochasia together forming the sister lineage of the remaining Utricularia species. Of the fundamental morphological specializations, the stoloniferous growth form apparently arose independently within Genlisea and Utricularia three times, and within Utricularia itself, perhaps more than once. The epiphytic habit has evolved independently at least three times, in Pinguicula, in Utricularia section Phyllaria, and within the two sections Orchidioides and Iperua (in the latter as bromeliad tank-epiphytes). The suspended aquatic habit may have evolved independently within sections Utricularia and Vesiculina. Biogeographic optimization on the phylogeny demonstrates patterns commonly associated with the boreotropics hypothesis and limits the spatial origin of Lentibulariaceae to temperate Eurasia or tropical America.

The phylogeography and connectivity of the latitudinally widespread scleractinian coral Plesiastrea versipora in the Western Pacific

Whereas terrestrial animal populations might show genetic connectivity within a continent, marine species, such as hermatypic corals, may have connectivity stretching to all corners of the planet. We quantified the genetic variability within and among populations of the widespread scleractinian coral, Plesiastrea versipora along the eastern Australian seaboard (4145 km) and the Ryukyu Archipelago (Japan, 681 km) using sequences of internal transcribed spacers (ITS1-2) from ribosomal DNA. Geographic patterns in genetic variability were deduced from a nested clade analysis (NCA) performed on a parsimony network haplotype. This analysis allowed the establishment of geographical associations in the distribution of haplotypes within the network cladogram, therefore allowing us to deduce phylogeographical patterns based under models of restricted gene flow, fragmentation and range expansion. No significant structure was found among Ryukyu Archipelago populations. The lack of an association between the positions of haplotypes in the cladogram with geographical location of these populations may be accounted for by a high level of gene flow of P. versipora within this region, probably due to the strong Kuroshio Current. In contrast, strong geographical associations were apparent among populations of P. versipora along the south-east coast of Australia. This pattern of restricted genetic connectivity among populations of P. versipora on the eastern seaboard of Australia seems to be associated with the present surface ocean current (the East Australian Current) on this side of the south-western Pacific Ocean.

Phylogeny of the Sporobolus indicus complex, based on internal transcribed spacer (ITS) sequences

The entire internal transcribed spacer ( ITS) region, including the 5.8S subunit of the nuclear ribosomal DNA ( rDNA), was sequenced by direct double-stranded sequencing of polymerase chain reaction (PCR) amplified fragments. The study included 40 Sporobolus ( Family Poaceae, subfamily Chloridoideae) seed collections from 14 putative species ( all 11 species from the S. indicus complex and three Australian native species). These sequences, along with those from two out-group species [ Pennisetum alopecuroides ( L.) Spreng. and Heteropogon contortus ( L.) P. Beauv. ex Roemer & Schultes, Poaceae, subfamily Panicoideae], were analysed by the parsimony method (PAUP; version 4.0b4a) to infer phylogenetic relationships among these species. The length of the ITS1, 5.8S subunit and ITS2 region were 222, 164 and 218 base pairs ( bp), respectively, in all species of the S. indicus complex, except for the ITS2 region of S. diandrus P. Beauv. individuals, which was 217 bp long. Of the 624 characters included in the analysis, 245 ( 39.3%) of the 330 variable sites contained potential phylogenetic information. Differences in sequences among the members of the S. pyramidalis P. Beauv., S. natalensis (Steud.) Dur & Schinz and S. jacquemontii Kunth. collections were 0%, while differences ranged from 0 to 2% between these and other species of the complex. Similarly, differences in sequences among collections of S. laxus B. K. Simon, S. sessilis B. K. Simon, S. elongatus R. Br. and S. creber De Nardi were 0%, compared with differences of 1-2% between these four species and the rest of the complex. When comparing S. fertilis ( Steud.) Clayton and S. africanus (Poir.) Robyns & Tourney, differences between collections ranged from 0 to 1%. Parsimony analysis grouped all 11 species of the S. indicus complex together, indicating a monophyletic origin. For the entire data set, pair-wise distances among members of the S. indicus complex varied from 0.00 to 1.58%, compared with a range of 20.08-21.44% among species in the complex and the Australian native species studied. A strict consensus phylogenetic tree separated 11 species of the S. indicus complex into five major clades. The phylogeny, based on ITS sequences, was found to be congruent with an earlier study on the taxonomic relationship of the weedy Sporobolus grasses revealed from random amplified polymorphic DNA ( RAPD). However, this cladistic analysis of the complex was not in agreement with that created on past morphological analyses and therefore gives a new insight into the phylogeny of the S. indicus complex.

A review of the genus Ledermuelleriopsis Willmann (Acari : Prostigmata : Stigmaeidae)

The mite family Stigmaeidae (Acari:Prostigmata) is of considerable importance in biological control, but its genera are often poorly defined and have never been subjected to cladistic analysis. Herein, we report the stigmaeid genus Ledermuelleriopsis Willmann from Australia for the first time, present a preliminary phylogenetic analysis that demonstrates that Eustigmaeus Berlese and Ledermuelleriopsis Willman are distinct, review the genus at the world level, and provide diagnostic characters of the adult females for each of the 21 known species. We also catalogue habitats, distributions and localities of holotypes. Four new species from Australia are described and illustrated: L. parvilla, sp. nov. from old dune sand, L. barbellata, sp. nov. from wet-sandy heath litter, and L. pustulosa, sp. nov. and L. claviseta, sp. nov. from dry eucalypt forest litter. A key to adult females of all known Ledermuelleriopsis species is provided. The Australian species and L. incisa Wood from New Zealand can be separated from all other members of the genus by a synapomorphy: the reduction of the number of setae on the aggenital shield to one pair. Results of a preliminary morphological cladistic analysis for those stigmaeid genera in which the larvae and adults of both sexes are known, indicate that Ledermuelleriopsis is basal to a clade containing Cheylostigmaeus Willman and Eustigmaeus.

An evolutionary radiation of beeflies in semi-arid Australia: systematics of the Exoprosopini (Diptera : Bombyliidae)

Almost half of the 4822 described beeflies in the world belong to the subfamily Anthracinae, with most of the diversity found in three cosmopolitan tribes: Villini, Anthracini, and Exoprosopini. The Australian Exoprosopini previously contained three genera, Ligyra Newman, Pseudopenthes Roberts and Exoprosopa Macquart. Pseudopenthes is an Australian endemic, with two species including Ps. hesperis, sp. nov. from Western Australia. Two new species of the exoprosopine Atrichochira Hesse, Atr. commoni, sp. nov. and Atr. paramonovi, sp. nov., are also described from Australia, extending the generic distribution from Africa. Cladistic analysis clarified the phylogenetic relationships between the recognised groups of the Exoprosopini and determined generic limits on a world scale. Inclusion of 18 Australian exoprosopines placed the Australian species in the context of the world fauna. The Exoprosopini contains six large groups. The basal group I contains species previously included in Exoprosopa to which the name Defilippia Lioy is applied. Group II contains Heteralonia Rondani, Atrichochira, Micomitra Bowden, Pseudopenthes, and Diatropomma Bowden. Colossoptera Hull is newly synonymised with Heteralonia. Group III is a paraphyletic assemblage of Pterobates Bezzi and Exoprosopa including the Australian Ex. sylvana ( Fabricius). Ligyra is paraphyletic, forming two well-separated clades. The African clade is described as Euligyra Lambkin, gen. nov., which, together with Litorhina Bezzi and Hyperalonia Rondani, form group IV. The Australian group V is true Ligyra. The remaining monophyletic lineage of exoprosopines, group VI, the Balaana-group of genera, shows evidence of an evolutionary radiation of beeflies in semi-arid Australia. Phylogenetic analysis of all 42 species of the Balaana-group of genera formed a basis for delimiting genera. Seven new genera are described by Lambkin & Yeates: Balaana, Kapua, Larrpana, Munjua, Muwarna, Palirika and Wurda. Four non-Australian species belong to Balaana. Thirty two new Australian species are described: Bal. abscondita, Bal. bicuspis, Bal. centrosa, Bal. gigantea, Bal. kingcascadensis, K. corusca, K. irwini, K. westralica, Lar. collessi, Lar. zwicki, Mun. erugata, Mun. lepidokingi, Mun. paralutea, Mun. trigona, Muw. vitreilinearis, Pa. anaxios, Pa. basilikos, Pa. blackdownensis, Pa. bouchardi, Pa. cyanea, Pa. danielsi, Pa. decora, Pa. viridula, Pa. whyalla, W. emu, W. impatientis, W. montebelloensis, W. norrisi, W. patrellia, W. skevingtoni, W. windorah, and W. wyperfeldensis. The following new combinations are proposed: from Colossoptera: Heteralonia latipennis (Brunetti); from Exoprosopa: Bal. grandis (Pallas), Bal. efflatounbeyi (Paramonov), Bal. latelimbata ( Bigot), Bal. obliquebifasciata ( Macquart), Bal. tamerlan (Portschinsky), Bal. onusta ( Walker), Def. busiris (Jaennicke), Def. efflatouni ( Bezzi), Def. eritreae (Greathead), Def. gentilis ( Bezzi), Def. luteicosta ( Bezzi), Def. minos (Meigen), Def. nigrifimbriata ( Hesse), Def. rubescens ( Bezzi), K. adelaidica ( Macquart), Lar. dimidiatipennis ( Bowden), Muw. stellifera ( Walker), and Pa. marginicollis ( Gray); from Ligyra: Eu. enderleini ( Paramonov), Eu. mars ( Bezzi), Eu. monacha (Klug), Eu. paris ( Bezzi), Eu. sisyphus ( Fabricius), and Eu. venus (Karsch).

Phylogeography of an east Australian wet-forest bird, the satin bowerbird (Ptilonorhynchus violaceus), derived from mtDNA, and its relationship to morphology

Australian wet forests have undergone a contraction in range since the mid-Tertiary, resulting in a fragmented distribution along the east Australian coast incorporating several biogeographical barriers. Variation in mitochondrial DNA and morphology within the satin bowerbird was used to examine biogeographical structure throughout almost the entire geographical extent of these wet forest fragments. We used several genetic analysis techniques, nested clade and barrier analyses, that use patterns inherent in the data to describe the spatial structuring. We also examined the validity of the two previously described satin bowerbird subspecies that are separated by well-defined biogeographical barriers and tested existing hypotheses that propose divergence occurs within each subspecies across two other barriers, the Black Mountain corridor and the Hunter Valley. Our data showed that the two subspecies were genetically and morphologically divergent. The northern subspecies, found in the Wet Tropics region of Queensland, showed little divergence across the Black Mountain corridor, a barrier found to be significant in other Wet Tropics species. Biogeographical structure was found through southeastern Australia; three geographically isolated populations showed genetic differentiation, although minimal divergence was found across the proposed Hunter Valley barrier. A novel barrier was found separating inland and coastal populations in southern New South Wales. Little morphological divergence was observed within subspecies, bar a trend for birds to be larger in the more southerly parts of the species' range. The results from both novel and well-established genetic analyses were similar, providing greater confidence in the conclusions about spatial divergence and supporting the validity of these new techniques.