Cpdna rbcL sequences for Biodiverse analyses Final Report

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cpDNA rbcL sequences for Biodiverse analyses

Final Report

Prepared for the Ministry for the Environment by Dr Peter Heenan

Allan Herbarium (Landcare Research)


This report has been prepared by Landcare Research for Ministry for the Environment. If used by other parties, no warranty or representation is given as to its accuracy and no liability is accepted for loss or damage arising directly or indirectly from reliance on the information in it.
This report may be cited as:

Ministry of the Environment. 2015. cpDNA rbcL sequences for Biodiverse analyses: Final Report. Wellington: Ministry for the Environment.

Published in October 2015 by the
Ministry for the Environment
Manatū Mō Te Taiao
PO Box 10362, Wellington 6143, New Zealand

ISBN: 978-0-908339-13-6 (electronic)

Publication number: ME 1218

© Crown copyright New Zealand [Year]

This document is available on the Ministry for the Environment’s website:

1Aim 4

2Background 4

3Work completed 4

4We obtained samples representing 211 indigenous New Zealand genera from dried collections in the Allan Herbarium or fresh collections from cultivated material. These 211 genera represented 95% of the 222 genera that were represented by surrogates in the Biodiverse study. 4

5Herbarium vouchers information is presented in Appendix 1. 4

6For 118 samples we did not obtain sequences as we obtained no PCR product, weak PCR product, or the sequencing was messy. 4

7For some species we included more than one sample as we attempted multiple DNA extractions. 4

8Using a robot for DNA extractions has meant we were able to increase the sample number analysed, so we have been able to send three plates (each with c. 95 samples) for sequencing, rather than the two plates we initially envisaged. This has meant that for some samples that were unsuccessful in plate 1 (or plate 2) we were able to attempt another DNA extraction, PCR and sequence with a different sample in plates 2 and/or 3. For some genera we have attempted sequencing up to three different samples, and this has meant we have been able to obtain sequences representing more genera. 4

9For some samples identified as being particularly difficult, we attempted DNA extractions using a mortar & pestle and altered PCR protocols to obtain suitable DNA product for sequencing. 4

10Appendix 1 provides a summary of the successful sequence results, including GenBank numbers. 5

11The total dataset comprised 436 genera and was aligned in MEGA 5.0 (Tamura et al. 2011). A model of sequence evolution for rbcL was selected using ModelTest. 5

12An optimised Maximum Likelihood tree was used as the base tree for model likelihood calculations and the best model of sequence substitution was selected using the Bayesian information criterion. Bayesian inference of phylogeny was performed using MrBayes version 3.2.3 through the CIPRES Science Gateway version 3.3. Two runs with eight chains and a sample frequency of 5000 were run for 36,000,000 generations resulting in a total of 7200 trees for each run. The first 6000 trees of each run were discarded as burn-in and the remaining 2400 trees of both runs were combined in a 95% majority rule consensus tree using SumTrees version 3.3.1. The consensus tree is presented in Appendix 2. 5

13The Biodiverse software package version 1.0 was used for all analyses (Laffan et al., 2010). Spatial data used for this study comprised 213,141 georeferenced specimens from the New Zealand Virtual Herbarium (NZVH). All analyses were performed using a cell size of 0.12°, resulting in 2393 cells. 5

14The genus-level spatial data and phylogenetic tree were used to calculate phylogenetic diversity (PD) and phylogenetic corrected weighted endemism (PE_CWE) for the entire New Zealand archipelago and the main New Zealand islands. Statistical significance of the resulting patterns of endemism for each of the phylogenetic and non-phylogenetic analyses was assessed with a two-tailed test involving 999 random realisations of the observed datasets using the preserved model implemented in Biodiverse. 5

15Categorical Analyses of Neo- and Palaeo- Endemism (CANAPE) analyses. Phylogenetic diversity (PD) and phylogenetic weighted endemism (PWE) were calculated following Mishler et al. (2014) at genus and species rank. Statistical significance of the resulting biodiversity patterns for PD and PWE was assessed with 999 random realisations of the observed datasets using the preserved model implemented in Biodiverse. This model randomises the spatial locations of each taxon while preserving the taxon range and maintaining the taxon richness within each cell. 5

16A CANAPE analysis was performed on the genus and species spatial and phylogenetic data following Mishler et al. (2014). Differences in neo- and palaeo-endemism among islands were visualised as barplots by plotting the distribution of p(RPE) values for the entire New Zealand archipelago, North Island, South Island and offshore islands. 5

17Spatial and phylogenetic analyses results and discussion 6

18For the 31 genera for which we have not obtained DNA sequences from New Zealand indigenous species, this missing data should be obtained to complete the dataset of rbcL sequences representative of all New Zealand genera. This is achievable and loans from other herbaria with recent collections of these genera and some field work is required to obtain suitable plant material for sequencing. It should be noted that the orchids Gastrodia and Molloybas and the parasitic Dactylanthus do not have chlorophyll and therefore are not able to be sequenced for rbcL. 8

19Having almost completed the construction of an rbcL phylogeny for New Zealand indigenous vascular plant genera, consideration should be given to expanding this to include indigenous non-vascular moss, liverwort and hornwort genera (bryophytes). 8

20Discussions have been held with Dr Robbie Holdaway (Landcare Research) about utilising the New Zealand indigenous genus rbcL phylogeny as part of the environmental DNA project (A national framework for biological heritage assessment across natural and productive landscapes) in The National Science Challenge, New Zealand’s Biological Heritage. Completing the rbcL sequencing for the missing genera is a priority as the resulting dataset would have multiple uses. 8

21References 8

Appendix 1. Sample information for the new rbcL sequences generated for this contract 9

Appendix 2. Genus rbcL phylogeny 15

Appendix 3. Generic phylogenetic endemism 16

Appendix 4. Genus-level patterns of PD, PWE and neo- and palaeo-endemism 17

Appendix 5. Species-level patterns of PD, PWE and neo- and palaeo-endemism 18

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