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Congruence of community structure between taxonomic identification and T-RFLP analyses in free-living soil nematodes
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Congruence of community structure between taxonomic identification and T-RFLP analyses in free-living soil nematodes Paul B.L. George ∗ , Zoë Lindo The University of Western Ontario, Department of Biology, London, Ontario, Canada N6A 5B7
a r t i c l e
i n f o
Article history: Received 6 February 2015 Received in revised form 22 April 2015 Accepted 22 April 2015 Keywords: T-RFLP Nematodes Community composition Similarity matrices
a b s t r a c t Molecular-based methods of community analysis are becoming a popular alternative to the traditional, highly specialized, and time-consuming taxonomic identifications especially for morphologically challenging groups like free-living soil nematodes. In particular, terminal restriction fragment length polymorphism (T-RFLP) analysis has become popular as a quick and efficient tool to provide researchers with broad-scale assessments of community structure. The majority of studies comparing T-RFLP with morphological-based assessments have used cultured or previously characterized communities. Here we compare morphological identification to T-RFLP analyses in a previously unexplored system for nematode diversity, the boreal forest of Ontario, Canada. Samples were collected from five silvicultural treatments over two sampling seasons with nematodes extracted using the Baermann funnel technique. We found significantly greater richness from T-RFLP analyses than morphological identifications at both sampling times, but in both cases silvicultural treatment had no effect. Despite differences in taxonomic richness, community compositional similarities were highly correlated between morphological and molecular methods for both sampling times. We suggest that both methods are reliable in studies of previously undescribed communities where the goal is community assessment under treatment. © 2015 Elsevier GmbH. All rights reserved.
Assessments for composition of free-living nematode community structures are critical for quantifying community change. These animals have been recognized as good indicators of soil quality (Neher 2001) due to their high abundances (Yeates et al. 2009), trophic and functional diversity (Bongers and Bongers 1998), as well as their ease of sampling (Ferris et al. 2001). Indeed this has been well-documented through the use of trait-based indices (e.g. Maturity Index) for the assessment of the responses of a nematode community to anthropogenic disturbance (Bongers et al.,1990; Ferris et al., 2001; George and Lindo 2015). For example, the effects of pollution, fertilization, and ecosystem management have been extensively characterized using nematode community structure (Bongers 1999). Trait-based approaches are based on morphological assessment and knowledge of biology, which may not be feasible for researchers lacking experience with nematodes, as these animals require extensive taxonomic expertise (Coomans 2002) and time to identify (George and Lindo 2015). In fact, as the time required to identify this incredibly abundant taxon is so great, generally only the first 100–200 individuals in a sample are identified (Bongers 1994). Combined, these two limitations may
∗ Corresponding author. Tel.: +1 519 661 2111x82284; fax: +1 519 661 3935. E-mail address: [email protected] (P.B.L. George).
reduce the efficacy of conventional taxonomy-based assessments of nematode communities. Various methods have attempted to overcome such impairments, including strict trait-based approaches that can be used independently of taxonomic identity like body size (Turnbull et al. 2014; George and Lindo 2015), and molecular techniques. Indeed the use of molecular techniques to discern community structure has been aggressively explored (Wiesel et al. 2015). Polymerase chain reaction (PCR)-based techniques such as denaturing gradient gel electrophoresis (DGGE) (Foucher et al. 2004; Okada and Oba 2008), DNA barcoding/sequencing (Vervoort et al. 2012), and terminal restriction fragment length polymorphism (T-RFLP) (Donn et al. 2008; Donn et al. 2012; Lott et al. 2014; Wiesel et al. 2015) have been commonly used to address questions of community composition in difficult taxonomic groups (Chen et al. 2010). Terminal restriction fragment length polymorphism in particular holds much potential as it is a comparatively inexpensive method, the result of which can quickly be compared between different gel runs (Liu et al. 1997; Chen et al. 2010). The T-RFLP method combines PCR and restriction enzyme techniques to target and amplify sequences of DNA with a fluorescently labeled primer using DNA extracted from the whole community. The mixed PCR-product is exposed to a restriction enzyme, which digests the DNA, cutting it at target sites specific to the taxa of
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Please cite this article in press as: George, P.B.L., Lindo, Z., Congruence of community structure between taxonomic identification and T-RFLP analyses in free-living soil nematodes. Pedobiologia - J. Soil Ecol. (2015), http://dx.doi.org/10.1016/j.pedobi.2015.04.003
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concern. Each fluorescently labeled band produced through T-RFLP analyses represents a unique operational taxonomic unit (OTU) (Liu et al. 1997). Although each OTU lacks a species-level identification by definition, it is possible to assign such a label to them when the technique is conducted using known cultures (Liu et al. 1997; Chen et al. 2010) or through novel techniques that integrate sequencing of the DNA from specific individuals (Donn et al. 2012). T-RFLP methods have proven useful in cases where a previously (Lott et al. 2014) or concurrently (Donn et al. 2012) characterized reference community of nematodes exists and when novel methodologies are under examination (Donn et al. 2008; Edel-Hermann et al. 2008; Wiesel et al. 2015). Yet, has not been compared for the assessment of natural communities where abundance and richness values are previously unknown. Here we compare taxonomic richness derived from conventional morphological identification to the richness of OTUs produced from T-RFLP analyses using parallel sampling and analysis for each method. We use free-living soil nematode communities from five forest management treatments to assess the comparability/similarity of traditional identification and T-RFLP analyses at two sampling times. Twenty boreal forest soil samples were collected from the Island Lake Biomass Research and Demonstration Area near Chapleau, Ontario, Canada (47◦ 50 N 83◦ 24 W) in June and August 2013. We collected five replicate samples from each of the following silviculture treatment implemented in summer 2012: uncut forest, clear-cut forest, clear-cut with 100 kg/ha wood ash amendment, clear-cut with 200 kg/ha wood ash amendment, and clear-cut with 400 kg/ha wood ash amendment. Full site and treatment descriptions are given in Kwiation et al. (2011) and George and Lindo (2015). Samples were collected using core sampling (5 cm diameter × 15 cm in depth) and homogenized into two 25 g wet soil weight aliquots for morphological identification and T-RFLP analyses. Nematodes for both morphological and molecular assessments were extracted using the Baermann funnel technique (Forge and Kimpinski 2008). For morphological identification, nematodes were extracted into water over 72 hr, fixed with 4% formalin, and stained with Rose Bengal prior to identification and enumeration. Individuals were identified to morphotaxon at the family or genus level (Table 1) following slide-mounting in Permount medium using keys from Bongers (1994) and the University of Nebraska–Lincoln (Tarjan et al. 1977). For T-RFLP analyses, nematodes were extracted into water and frozen at -80 C until DNA extraction. Frozen molecular samples were thawed at room temperature and DNA extracted using bead-beating in conjunction with PureLink® genomic DNA extraction kits. The DNA was purified using a Zymo DNA clean and concentrator kit® . Samples were processed with PCR using the forward primer Nem SSU F74 (5 AARCYGCGWAHRGCTCRKTA 3 ) with the fluorescent label 6fluorescein amidite (6-FAM), the reverse primer SSU R 81 (5 TGATCCWKCYGCAGGTTCAC 3 ) (Donn et al. 2011), and AccuStart II PCR ToughMix® . These reagents were combined with whole community DNA and nuclease-free water in a 25 L reaction with the following amounts: 12.5 L AccuStart II PCR ToughMix, 5 L nuclease-free water, 1.25 L forward primer, 1.25 L reverse primer (both at 20 pMolar concentration), and 5 L DNA template. A positive control for the PCR was derived from a commercial culture of Heterorhabditis bacteriophora (Poinar) and Steinernema carpocapsae (Weiser). The PCR reaction was conducted following Donn et al. (2011): 94 ◦ C, 2 min; then 35 cycles of 94 ◦ C, 30 s; 51 ◦ C, 1 min, 68 ◦ C, 2 min, and a final extension step of 68 ◦ C for 10 min. This process yielded products of approximately 1750 base pairs that were subsequently digested with Hinf1 restriction endonuclease (Donn et al. 2012) in a 32 L reaction consisting of: 10 L PCR reaction mixture, 18 L
A
Forest Clear-cut 100kg/ha wood ash 200kg/ha wood ash 400kg/ha wood ash
B
Forest Clear-cut 100kg/ha wood ash 200kg/ha wood ash 400kg/ha wood ash Fig. 1. Plots generated from non-metric dimensional scaling (NMDS) of the nematode community as assessed by (A) morphological identification (stress = 0.05) and (B) T-RFLP (stress = 0.11) analysis in June.
nuclease-free water, 2 L 10X buffer R, and 2 L Hinf1. The digestion products were sent to the Advanced Analysis Centre at the University of Guelph for processing using a 500 LIZ size standard and returned for further analyses. Restriction fragment analyses were conducted using GeneMarker (Softgenetics), which produces an output that displays bands as peaks. This allowed for the quantification of presence/absence data based on peaks. Differences between taxonomic and OTU richness were compared for each sampling time using a factorial analysis of variance (ANOVA) for silviculture treatments. To compare the community structure of taxonomic identification and T-RFLP, similarity matrices of community composition for each method at each sample time were created based on square-root Euclidean distance for morphological data and presence/absence Euclidean distance for OTU data using Primer 5 (Primer-E Ltd., 2001). Similarity matrices were generated and compared using a Mantel test with 9999 repetitions. Community compositions are presented as non-metric dimensional scaling (NMDS) analyses (Fig. 1), and silviculture treatments compared using analysis of similarity (ANOSIM) tests. Taxonomic identification yielded 26 morphotaxa total (22 June and 24 August). The most frequently observed groups were the Rhabditidae, Plectus sp., and Acrobeloides sp. (Table 1). Each genus was monotypic by morphology; however, there were three to four different morphs for each family-level identification. T-RFLP revealed 92 OTUs in June and 80 in August (174 total) that had greater than 60 base pairs. Factorial ANOVAs found significant differences between taxonomic and OTU richness at both sampling times, with OTU richness values greater than those derived from taxonomic identifications (June: F1,30 = 24.316, P < 0.001; August: F1,30 = 27.490, P < 0.001). However, there were no differences between richness based
Please cite this article in press as: George, P.B.L., Lindo, Z., Congruence of community structure between taxonomic identification and T-RFLP analyses in free-living soil nematodes. Pedobiologia - J. Soil Ecol. (2015), http://dx.doi.org/10.1016/j.pedobi.2015.04.003
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Table 1 Mean nematode abundances across all samples of each taxonomic group observed in June and August. Numbers in parenthesis are standard deviation. Taxonomic identity
Mean abundance
Family
Genus
June
August
Alaimidae
Alaimus sp. Paramphidelus sp. Sectonema sp. Bastiania Acrobeloides sp. Cephalobus sp. Chiloplacus sp. Eucephalobus sp. Criconema sp. Criconemoides sp. Macroposthonia sp. Hemicycliophora sp. Fungiotonchium sp. Clarkus sp. Paravulvus sp.
14.75 (±21.70) 10.00 (±16.55) 0.0 (±0.0) 0.0 (±0.0) 24.1 (±) 0.2 (±0.52) 0.05(±0.22) 0.4 (±0.99) 0.15 (±0.49) 0.05 (±0.22) 0.0 (±0.0) 1.75 (±2.94) 0.0 (±0.0) 1.65 (±1.84) 0.0 (±0.0) 13.95 (±17.42) 26.50 (±38.40) 12.95 (±24.85) 0.55 (±0.89) 0.25 (±0.55) 5.35 (±6.08) 1.65 (±1.84) 18.25 (±19.95) 9.5 (±15.92) 4.2 (±6.66) 0.05 (±0.22) 0.75 (±1.29) 25.2 (±36.56)
4.92 (±4.96) 5.47 (±5.58) 1.0 (±0) 2.6 (±1.52) 18.32 (±39.75) 12.15 (±12.32) 0.0 (±0.0) 8.0 (±10.80) 0.0 (±0.0) 0.0 (±0.0) 1.0 (±0) 3.06 (±1.92) 2.0 (±1.41) 2.38 (±1.94) 2.0 (±1.41) 6.22 (±4.24) 16.42 (±8.20) 4.06 (±3.38) 2.8 (±3.49) 2.2 (±2.17) 7.72 (±11.97) 2.46 (±1.45) 11.88 (±12.41) 2.85 (±2.08) 1.83 (±0.98) 0.0 (±0.0) 1.89 (±0.78) 10.47 (±11.03)
Aporcelaimidae Bastianiidae Cephalobidae
Criconematidae
Hemicycliophoridae Iotonchiidae Mononchidae Nygolaimidae Panagrolaimidaea Plectidae Prismatolaimidae Qudsianematidae
Rhabditidaea Teratocephalidae Tripylidae Tylopharyngidae Unknown a
Plectus sp. Wilsonema sp. Prismatolaimus sp. Epidorylaimus sp. Eudorylaimus sp. Thonus sp. Teratocephalus sp. Tripyla sp. Trischistoma sp. Tylolaimophorus sp.
Note: these families could not be consistently identified to genus level; however, three morphotypes were identified in the Panagrolaimidae and four in the Rhabditidae.
on silviculture treatment (June: F4,30 = 0.316, P = 0.865; August: F4,30 = 1.141, P = 0.356) nor the interaction of richness and treatment (June: F4,30 = 0.383, P = 0.819; August: F4,30 = 1.202, P = 0.330). A positive correlation of community similarity between methods was present in both June and August. Although this trend was not statistically significant in June (R = 0.252, P = 0.061), it was in August (R = 0.299, P = 0.038), and both values are considered biologically relevant. The plots of community distribution for each either morphological or molecular data show generally conserved groupings of treatments, with low stress values (Fig. 1). The ANOSIM tests showed no significant groupings within treatments in morphological data at either sampling times (global R = −0.057, P = 0.804 and global R = −0.045, P = 0.713, in June and August, respectively) or OTU data (global R = 0.028, P = 0.341 and global R = 0.015, P = 0.368, in June and August, respectively). Although there were significant differences between the richness values for taxonomic identification and T-RFLP analyses, the resulting communities were largely similar. The high OTU richness compared to morphological richness values is not unexpected. The amplification of non-target sequences in the PCR step is a common result of many primers. While the primer combination we used is established for T-RFLP analyses of nematode communities (Donn et al. 2011, 2012), the forward primer (NEM SSU F74) is not specific to nematodes and capable of amplifying other eukaryotes including tardigrades, annelids, and various protists (Donn et al. 2011). The use of non-specific primers allows for recognition of the greatest amount of nematode diversity, however, this comes at the risk of amplifying non-target DNA, which can inflate nematode diversity. This can be mitigated to a certain degree by extracting nematodes from soil samples before DNA extraction takes place (Donn et al. 2011). Tardigrades were occasionally observed amongst nematodes extracted for morphological identification, which could have also been present with the nematodes extracted for DNA analyses and thereby, may have contributed to the increased number of OTUs.
Genus and family-level identification also potentially underestimates morphological richness. While genera were monotypic by morphology, there were three to four different morphs for each family level identification that could not be reliably grouped. Identification to family or genus is common for studies of nematode communities, largely due to the extreme difficulty involved in making species-level distinctions and identifications in areas where the nematode communities have not been previously described (Panesar and Marshall 2003). It should be noted; however, that in many studies of nematode communities from Canadian forests often only one species has been observed for a single genus (Sutherland 1965; Panesar et al. 2000, 2001). For families such as the Panagrolaimidae and Rhabditidae that are common in soils and may contain multiple genera (Sohlenius 2002), we observed three morpho-types within the Panagrolaimidae and four within the Rhabditidae that could never be consistently identified. Of all the nematodes enumerated in the present study, 13% were unidentifiable. These individuals were generally the smallest nematodes observed, and were likely larval stages, which are incredibly difficult to accurately identify (Chen et al. 2010). These individuals were enumerated for abundance, but not included in richness values. Other nematodes that could not be identified were generally obscured or damaged by debris, meaning they could not be accurately classified. The degree of taxonomic resolution likely only explains small differences between morphologic and molecular richness values. The presence of cryptic species may contribute to the higher OTU richness observed. Cryptic species, defined as sympatric populations that are genetically distinct yet retain a superficial morphological similarity and may be recently diverged or reproductively isolated, have recently been the focus of much debate in studies of global biodiversity (Bickford et al. 2007). For nematodes, many have been acknowledged as possessing cosmopolitan distributions (Bongers 1994), however, this is controversial. Nematodes fall into the size range demarcating the
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border between geographically restricted macrofauna and cosmopolitan microfauna (Finlay and Fenchel 2004). Indeed although there is evidence for a cosmopolitan distribution of some marine (Bik et al. 2010), freshwater (Abebe and Coomans 1995), and terrestrial nematodes (Zauner et al. 2007), biogeographic patterns of nematodes are still early in development (Zullini 2014) and cryptic species are continually being described in these groups (Jorge et al. 2013; Cavallero et al. 2014; Félix et al. 2014). Specifically, groups of cryptic species, or species complexes, have been defined for some of the most important and frequently encountered nematode taxa including common parasitic (Cavallero et al. 2014) and free-living lineages (Félix et al. 2014). Species complexes are present in some of the taxa identified here for example Eudorylaimus (Panesar et al. 2000) and members of the Rhabditidae (Sohlenius 2002). This suggests that difference between morphologic and molecular richness values may, in part, be due cryptic diversity. Despite differences in absolute richness values, these two methods produce similar community composition data, and treatment differences under an applied context. Foucher et al. (2004) demonstrated similar richness values from sub-samples analyzed by traditional taxonomic identification and DGGE. However, in that case richness was often underestimated by the DGGE method. Okada and Oba (2008) also show a similar, yet stronger correlation between taxonomic identification and DGGE methodologies. Although to our knowledge, this is the first explicit comparison of taxonomic identification and T-RFLP analyses in a natural system, it is encouraging that the results presented reflect other studies. Yet, it should be noted that the use of molecular methods, including T-RFLP, are not currently compatible with ecological fieldwork where interest is in understanding the relationships between nematode diversity and ecosystem function. Indeed, without taxonomic identities, the nematode community cannot be characterized using trait-based indices such as the Maturity Index. However, the use of coarse molecular methods like T-RFLP can be considered adequate indicators of community structure that can appropriately supplement traditional the traditional taxonomic approach or be used as a surrogate for that method in controlled settings when expertise is sufficiently lacking. Further development of T-RFLP peak databases collected from known communities will increase the efficiency of this community composition analyses. Similarly, profiles from sites characterized by trait-based indices may be able to accurately show changes in the local community over time. We strongly suggest such potential avenues be investigated as additional tools to study this important indicator community.
Acknowledgements This work is funded by the National Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant program to ZL. We thank the Canadian Forest Service – Sault Ste-Marie, especially Paul Hazlett, the Ontario Ministry of Natural Resources, Ontario Power, and Tembec as well as their partners, the Northeast Superior Regional Chief’s Forum and the Northeast Superior Forest Community for the use of their Island Lake Biomass Harvest Research and Demonstration Area.
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Short communication
Congruence of community structure between taxonomic identification and T-RFLP analyses in free-living soil nematodes Paul B.L. George ∗ , Zoë Lindo The University of Western Ontario, Department of Biology, London, Ontario, Canada N6A 5B7
a r t i c l e
i n f o
Article history: Received 6 February 2015 Received in revised form 22 April 2015 Accepted 22 April 2015 Keywords: T-RFLP Nematodes Community composition Similarity matrices
a b s t r a c t Molecular-based methods of community analysis are becoming a popular alternative to the traditional, highly specialized, and time-consuming taxonomic identifications especially for morphologically challenging groups like free-living soil nematodes. In particular, terminal restriction fragment length polymorphism (T-RFLP) analysis has become popular as a quick and efficient tool to provide researchers with broad-scale assessments of community structure. The majority of studies comparing T-RFLP with morphological-based assessments have used cultured or previously characterized communities. Here we compare morphological identification to T-RFLP analyses in a previously unexplored system for nematode diversity, the boreal forest of Ontario, Canada. Samples were collected from five silvicultural treatments over two sampling seasons with nematodes extracted using the Baermann funnel technique. We found significantly greater richness from T-RFLP analyses than morphological identifications at both sampling times, but in both cases silvicultural treatment had no effect. Despite differences in taxonomic richness, community compositional similarities were highly correlated between morphological and molecular methods for both sampling times. We suggest that both methods are reliable in studies of previously undescribed communities where the goal is community assessment under treatment. © 2015 Elsevier GmbH. All rights reserved.
Assessments for composition of free-living nematode community structures are critical for quantifying community change. These animals have been recognized as good indicators of soil quality (Neher 2001) due to their high abundances (Yeates et al. 2009), trophic and functional diversity (Bongers and Bongers 1998), as well as their ease of sampling (Ferris et al. 2001). Indeed this has been well-documented through the use of trait-based indices (e.g. Maturity Index) for the assessment of the responses of a nematode community to anthropogenic disturbance (Bongers et al.,1990; Ferris et al., 2001; George and Lindo 2015). For example, the effects of pollution, fertilization, and ecosystem management have been extensively characterized using nematode community structure (Bongers 1999). Trait-based approaches are based on morphological assessment and knowledge of biology, which may not be feasible for researchers lacking experience with nematodes, as these animals require extensive taxonomic expertise (Coomans 2002) and time to identify (George and Lindo 2015). In fact, as the time required to identify this incredibly abundant taxon is so great, generally only the first 100–200 individuals in a sample are identified (Bongers 1994). Combined, these two limitations may
∗ Corresponding author. Tel.: +1 519 661 2111x82284; fax: +1 519 661 3935. E-mail address: [email protected] (P.B.L. George).
reduce the efficacy of conventional taxonomy-based assessments of nematode communities. Various methods have attempted to overcome such impairments, including strict trait-based approaches that can be used independently of taxonomic identity like body size (Turnbull et al. 2014; George and Lindo 2015), and molecular techniques. Indeed the use of molecular techniques to discern community structure has been aggressively explored (Wiesel et al. 2015). Polymerase chain reaction (PCR)-based techniques such as denaturing gradient gel electrophoresis (DGGE) (Foucher et al. 2004; Okada and Oba 2008), DNA barcoding/sequencing (Vervoort et al. 2012), and terminal restriction fragment length polymorphism (T-RFLP) (Donn et al. 2008; Donn et al. 2012; Lott et al. 2014; Wiesel et al. 2015) have been commonly used to address questions of community composition in difficult taxonomic groups (Chen et al. 2010). Terminal restriction fragment length polymorphism in particular holds much potential as it is a comparatively inexpensive method, the result of which can quickly be compared between different gel runs (Liu et al. 1997; Chen et al. 2010). The T-RFLP method combines PCR and restriction enzyme techniques to target and amplify sequences of DNA with a fluorescently labeled primer using DNA extracted from the whole community. The mixed PCR-product is exposed to a restriction enzyme, which digests the DNA, cutting it at target sites specific to the taxa of
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Please cite this article in press as: George, P.B.L., Lindo, Z., Congruence of community structure between taxonomic identification and T-RFLP analyses in free-living soil nematodes. Pedobiologia - J. Soil Ecol. (2015), http://dx.doi.org/10.1016/j.pedobi.2015.04.003
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concern. Each fluorescently labeled band produced through T-RFLP analyses represents a unique operational taxonomic unit (OTU) (Liu et al. 1997). Although each OTU lacks a species-level identification by definition, it is possible to assign such a label to them when the technique is conducted using known cultures (Liu et al. 1997; Chen et al. 2010) or through novel techniques that integrate sequencing of the DNA from specific individuals (Donn et al. 2012). T-RFLP methods have proven useful in cases where a previously (Lott et al. 2014) or concurrently (Donn et al. 2012) characterized reference community of nematodes exists and when novel methodologies are under examination (Donn et al. 2008; Edel-Hermann et al. 2008; Wiesel et al. 2015). Yet, has not been compared for the assessment of natural communities where abundance and richness values are previously unknown. Here we compare taxonomic richness derived from conventional morphological identification to the richness of OTUs produced from T-RFLP analyses using parallel sampling and analysis for each method. We use free-living soil nematode communities from five forest management treatments to assess the comparability/similarity of traditional identification and T-RFLP analyses at two sampling times. Twenty boreal forest soil samples were collected from the Island Lake Biomass Research and Demonstration Area near Chapleau, Ontario, Canada (47◦ 50 N 83◦ 24 W) in June and August 2013. We collected five replicate samples from each of the following silviculture treatment implemented in summer 2012: uncut forest, clear-cut forest, clear-cut with 100 kg/ha wood ash amendment, clear-cut with 200 kg/ha wood ash amendment, and clear-cut with 400 kg/ha wood ash amendment. Full site and treatment descriptions are given in Kwiation et al. (2011) and George and Lindo (2015). Samples were collected using core sampling (5 cm diameter × 15 cm in depth) and homogenized into two 25 g wet soil weight aliquots for morphological identification and T-RFLP analyses. Nematodes for both morphological and molecular assessments were extracted using the Baermann funnel technique (Forge and Kimpinski 2008). For morphological identification, nematodes were extracted into water over 72 hr, fixed with 4% formalin, and stained with Rose Bengal prior to identification and enumeration. Individuals were identified to morphotaxon at the family or genus level (Table 1) following slide-mounting in Permount medium using keys from Bongers (1994) and the University of Nebraska–Lincoln (Tarjan et al. 1977). For T-RFLP analyses, nematodes were extracted into water and frozen at -80 C until DNA extraction. Frozen molecular samples were thawed at room temperature and DNA extracted using bead-beating in conjunction with PureLink® genomic DNA extraction kits. The DNA was purified using a Zymo DNA clean and concentrator kit® . Samples were processed with PCR using the forward primer Nem SSU F74 (5 AARCYGCGWAHRGCTCRKTA 3 ) with the fluorescent label 6fluorescein amidite (6-FAM), the reverse primer SSU R 81 (5 TGATCCWKCYGCAGGTTCAC 3 ) (Donn et al. 2011), and AccuStart II PCR ToughMix® . These reagents were combined with whole community DNA and nuclease-free water in a 25 L reaction with the following amounts: 12.5 L AccuStart II PCR ToughMix, 5 L nuclease-free water, 1.25 L forward primer, 1.25 L reverse primer (both at 20 pMolar concentration), and 5 L DNA template. A positive control for the PCR was derived from a commercial culture of Heterorhabditis bacteriophora (Poinar) and Steinernema carpocapsae (Weiser). The PCR reaction was conducted following Donn et al. (2011): 94 ◦ C, 2 min; then 35 cycles of 94 ◦ C, 30 s; 51 ◦ C, 1 min, 68 ◦ C, 2 min, and a final extension step of 68 ◦ C for 10 min. This process yielded products of approximately 1750 base pairs that were subsequently digested with Hinf1 restriction endonuclease (Donn et al. 2012) in a 32 L reaction consisting of: 10 L PCR reaction mixture, 18 L
A
Forest Clear-cut 100kg/ha wood ash 200kg/ha wood ash 400kg/ha wood ash
B
Forest Clear-cut 100kg/ha wood ash 200kg/ha wood ash 400kg/ha wood ash Fig. 1. Plots generated from non-metric dimensional scaling (NMDS) of the nematode community as assessed by (A) morphological identification (stress = 0.05) and (B) T-RFLP (stress = 0.11) analysis in June.
nuclease-free water, 2 L 10X buffer R, and 2 L Hinf1. The digestion products were sent to the Advanced Analysis Centre at the University of Guelph for processing using a 500 LIZ size standard and returned for further analyses. Restriction fragment analyses were conducted using GeneMarker (Softgenetics), which produces an output that displays bands as peaks. This allowed for the quantification of presence/absence data based on peaks. Differences between taxonomic and OTU richness were compared for each sampling time using a factorial analysis of variance (ANOVA) for silviculture treatments. To compare the community structure of taxonomic identification and T-RFLP, similarity matrices of community composition for each method at each sample time were created based on square-root Euclidean distance for morphological data and presence/absence Euclidean distance for OTU data using Primer 5 (Primer-E Ltd., 2001). Similarity matrices were generated and compared using a Mantel test with 9999 repetitions. Community compositions are presented as non-metric dimensional scaling (NMDS) analyses (Fig. 1), and silviculture treatments compared using analysis of similarity (ANOSIM) tests. Taxonomic identification yielded 26 morphotaxa total (22 June and 24 August). The most frequently observed groups were the Rhabditidae, Plectus sp., and Acrobeloides sp. (Table 1). Each genus was monotypic by morphology; however, there were three to four different morphs for each family-level identification. T-RFLP revealed 92 OTUs in June and 80 in August (174 total) that had greater than 60 base pairs. Factorial ANOVAs found significant differences between taxonomic and OTU richness at both sampling times, with OTU richness values greater than those derived from taxonomic identifications (June: F1,30 = 24.316, P < 0.001; August: F1,30 = 27.490, P < 0.001). However, there were no differences between richness based
Please cite this article in press as: George, P.B.L., Lindo, Z., Congruence of community structure between taxonomic identification and T-RFLP analyses in free-living soil nematodes. Pedobiologia - J. Soil Ecol. (2015), http://dx.doi.org/10.1016/j.pedobi.2015.04.003
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Table 1 Mean nematode abundances across all samples of each taxonomic group observed in June and August. Numbers in parenthesis are standard deviation. Taxonomic identity
Mean abundance
Family
Genus
June
August
Alaimidae
Alaimus sp. Paramphidelus sp. Sectonema sp. Bastiania Acrobeloides sp. Cephalobus sp. Chiloplacus sp. Eucephalobus sp. Criconema sp. Criconemoides sp. Macroposthonia sp. Hemicycliophora sp. Fungiotonchium sp. Clarkus sp. Paravulvus sp.
14.75 (±21.70) 10.00 (±16.55) 0.0 (±0.0) 0.0 (±0.0) 24.1 (±) 0.2 (±0.52) 0.05(±0.22) 0.4 (±0.99) 0.15 (±0.49) 0.05 (±0.22) 0.0 (±0.0) 1.75 (±2.94) 0.0 (±0.0) 1.65 (±1.84) 0.0 (±0.0) 13.95 (±17.42) 26.50 (±38.40) 12.95 (±24.85) 0.55 (±0.89) 0.25 (±0.55) 5.35 (±6.08) 1.65 (±1.84) 18.25 (±19.95) 9.5 (±15.92) 4.2 (±6.66) 0.05 (±0.22) 0.75 (±1.29) 25.2 (±36.56)
4.92 (±4.96) 5.47 (±5.58) 1.0 (±0) 2.6 (±1.52) 18.32 (±39.75) 12.15 (±12.32) 0.0 (±0.0) 8.0 (±10.80) 0.0 (±0.0) 0.0 (±0.0) 1.0 (±0) 3.06 (±1.92) 2.0 (±1.41) 2.38 (±1.94) 2.0 (±1.41) 6.22 (±4.24) 16.42 (±8.20) 4.06 (±3.38) 2.8 (±3.49) 2.2 (±2.17) 7.72 (±11.97) 2.46 (±1.45) 11.88 (±12.41) 2.85 (±2.08) 1.83 (±0.98) 0.0 (±0.0) 1.89 (±0.78) 10.47 (±11.03)
Aporcelaimidae Bastianiidae Cephalobidae
Criconematidae
Hemicycliophoridae Iotonchiidae Mononchidae Nygolaimidae Panagrolaimidaea Plectidae Prismatolaimidae Qudsianematidae
Rhabditidaea Teratocephalidae Tripylidae Tylopharyngidae Unknown a
Plectus sp. Wilsonema sp. Prismatolaimus sp. Epidorylaimus sp. Eudorylaimus sp. Thonus sp. Teratocephalus sp. Tripyla sp. Trischistoma sp. Tylolaimophorus sp.
Note: these families could not be consistently identified to genus level; however, three morphotypes were identified in the Panagrolaimidae and four in the Rhabditidae.
on silviculture treatment (June: F4,30 = 0.316, P = 0.865; August: F4,30 = 1.141, P = 0.356) nor the interaction of richness and treatment (June: F4,30 = 0.383, P = 0.819; August: F4,30 = 1.202, P = 0.330). A positive correlation of community similarity between methods was present in both June and August. Although this trend was not statistically significant in June (R = 0.252, P = 0.061), it was in August (R = 0.299, P = 0.038), and both values are considered biologically relevant. The plots of community distribution for each either morphological or molecular data show generally conserved groupings of treatments, with low stress values (Fig. 1). The ANOSIM tests showed no significant groupings within treatments in morphological data at either sampling times (global R = −0.057, P = 0.804 and global R = −0.045, P = 0.713, in June and August, respectively) or OTU data (global R = 0.028, P = 0.341 and global R = 0.015, P = 0.368, in June and August, respectively). Although there were significant differences between the richness values for taxonomic identification and T-RFLP analyses, the resulting communities were largely similar. The high OTU richness compared to morphological richness values is not unexpected. The amplification of non-target sequences in the PCR step is a common result of many primers. While the primer combination we used is established for T-RFLP analyses of nematode communities (Donn et al. 2011, 2012), the forward primer (NEM SSU F74) is not specific to nematodes and capable of amplifying other eukaryotes including tardigrades, annelids, and various protists (Donn et al. 2011). The use of non-specific primers allows for recognition of the greatest amount of nematode diversity, however, this comes at the risk of amplifying non-target DNA, which can inflate nematode diversity. This can be mitigated to a certain degree by extracting nematodes from soil samples before DNA extraction takes place (Donn et al. 2011). Tardigrades were occasionally observed amongst nematodes extracted for morphological identification, which could have also been present with the nematodes extracted for DNA analyses and thereby, may have contributed to the increased number of OTUs.
Genus and family-level identification also potentially underestimates morphological richness. While genera were monotypic by morphology, there were three to four different morphs for each family level identification that could not be reliably grouped. Identification to family or genus is common for studies of nematode communities, largely due to the extreme difficulty involved in making species-level distinctions and identifications in areas where the nematode communities have not been previously described (Panesar and Marshall 2003). It should be noted; however, that in many studies of nematode communities from Canadian forests often only one species has been observed for a single genus (Sutherland 1965; Panesar et al. 2000, 2001). For families such as the Panagrolaimidae and Rhabditidae that are common in soils and may contain multiple genera (Sohlenius 2002), we observed three morpho-types within the Panagrolaimidae and four within the Rhabditidae that could never be consistently identified. Of all the nematodes enumerated in the present study, 13% were unidentifiable. These individuals were generally the smallest nematodes observed, and were likely larval stages, which are incredibly difficult to accurately identify (Chen et al. 2010). These individuals were enumerated for abundance, but not included in richness values. Other nematodes that could not be identified were generally obscured or damaged by debris, meaning they could not be accurately classified. The degree of taxonomic resolution likely only explains small differences between morphologic and molecular richness values. The presence of cryptic species may contribute to the higher OTU richness observed. Cryptic species, defined as sympatric populations that are genetically distinct yet retain a superficial morphological similarity and may be recently diverged or reproductively isolated, have recently been the focus of much debate in studies of global biodiversity (Bickford et al. 2007). For nematodes, many have been acknowledged as possessing cosmopolitan distributions (Bongers 1994), however, this is controversial. Nematodes fall into the size range demarcating the
Please cite this article in press as: George, P.B.L., Lindo, Z., Congruence of community structure between taxonomic identification and T-RFLP analyses in free-living soil nematodes. Pedobiologia - J. Soil Ecol. (2015), http://dx.doi.org/10.1016/j.pedobi.2015.04.003
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border between geographically restricted macrofauna and cosmopolitan microfauna (Finlay and Fenchel 2004). Indeed although there is evidence for a cosmopolitan distribution of some marine (Bik et al. 2010), freshwater (Abebe and Coomans 1995), and terrestrial nematodes (Zauner et al. 2007), biogeographic patterns of nematodes are still early in development (Zullini 2014) and cryptic species are continually being described in these groups (Jorge et al. 2013; Cavallero et al. 2014; Félix et al. 2014). Specifically, groups of cryptic species, or species complexes, have been defined for some of the most important and frequently encountered nematode taxa including common parasitic (Cavallero et al. 2014) and free-living lineages (Félix et al. 2014). Species complexes are present in some of the taxa identified here for example Eudorylaimus (Panesar et al. 2000) and members of the Rhabditidae (Sohlenius 2002). This suggests that difference between morphologic and molecular richness values may, in part, be due cryptic diversity. Despite differences in absolute richness values, these two methods produce similar community composition data, and treatment differences under an applied context. Foucher et al. (2004) demonstrated similar richness values from sub-samples analyzed by traditional taxonomic identification and DGGE. However, in that case richness was often underestimated by the DGGE method. Okada and Oba (2008) also show a similar, yet stronger correlation between taxonomic identification and DGGE methodologies. Although to our knowledge, this is the first explicit comparison of taxonomic identification and T-RFLP analyses in a natural system, it is encouraging that the results presented reflect other studies. Yet, it should be noted that the use of molecular methods, including T-RFLP, are not currently compatible with ecological fieldwork where interest is in understanding the relationships between nematode diversity and ecosystem function. Indeed, without taxonomic identities, the nematode community cannot be characterized using trait-based indices such as the Maturity Index. However, the use of coarse molecular methods like T-RFLP can be considered adequate indicators of community structure that can appropriately supplement traditional the traditional taxonomic approach or be used as a surrogate for that method in controlled settings when expertise is sufficiently lacking. Further development of T-RFLP peak databases collected from known communities will increase the efficiency of this community composition analyses. Similarly, profiles from sites characterized by trait-based indices may be able to accurately show changes in the local community over time. We strongly suggest such potential avenues be investigated as additional tools to study this important indicator community.
Acknowledgements This work is funded by the National Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant program to ZL. We thank the Canadian Forest Service – Sault Ste-Marie, especially Paul Hazlett, the Ontario Ministry of Natural Resources, Ontario Power, and Tembec as well as their partners, the Northeast Superior Regional Chief’s Forum and the Northeast Superior Forest Community for the use of their Island Lake Biomass Harvest Research and Demonstration Area.
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Please cite this article in press as: George, P.B.L., Lindo, Z., Congruence of community structure between taxonomic identification and T-RFLP analyses in free-living soil nematodes. Pedobiologia - J. Soil Ecol. (2015), http://dx.doi.org/10.1016/j.pedobi.2015.04.003